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I Marchetti

Nicolas Sari-Minodier

P Molinari

Chanez

Le ´a Touri

He ´le `ne Marchetti

Ire ` Ne Sari-Minodier

#," Nicolas Molinari

Pascal Chanez
email: pascal.chanez@univ-med.fr

The airport atmospheric environment: respiratory health at work

Keywords: Airport, air traffic, jet exhaust, occupational exposure, respiratory problems, respiratory tract

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he International Air Transport Association announced a slight decline in air traffic in May 2012 due to the economic environment, but air flight transport should continue to grow in the future as there is a trend to optimise time over shorter distances. Major hub airports are not big enough and are in need of a geographic expansion, which is often discussed. These changes cannot be made without taking into account the ''expansion'' of the adverse effects, both for workers who operate in the airport and for the surrounding neighbourhood. It is not sufficient only to discuss a landscape change or the risk of accident. Air pollution is a major concern for people situated at or near airports. These issues necessitate additional studies. The larger the airport, the stronger the impact on the population's health is [START_REF] Cohen | Air transportation and human health[END_REF]. Therefore, in this review, we discuss whether there is a correlation between atmospheric exposure at the airport, and exposure in the vicinity and occupational exposure (fig. 1).

AIRPORT ATMOSPHERIC ENVIRONMENT

Different research teams have focused on the characterisation of air pollution in the airport area and its surroundings. The concentration of particulate pollutants seems to be inversely proportional to the distance from the airport: the further away you are from the airport platform, the lower the pollution is [START_REF] Cohen | Airport-related air pollution and noise[END_REF]. Kerosene is the major compound in jet fuel and has a specific odour, especially before fuel combustion, which can be smelled .8 km from the airport [START_REF] Franssen | Assessing health consequences in an environmental impact assessment, the case of Amsterdam Airport Schiphol[END_REF].

The main polluting substances considered in this environment are nitrogen oxides, carbon dioxide, carbon monoxide, volatile organic compounds (VOCs) including polycyclic aromatic hydrocarbons (PAHs), sulfur dioxide [4], and fine and ultrafine particles (UFPs) [START_REF] Nemmar | Passage of inhaled particles into the blood circulation in humans[END_REF].

PAHs are dangerous. They have been measured and characterised for decades. Thus, it has been established that aromatic hydrocarbons are a significant part of jet exhaust pollution [START_REF] Cohen | Airport-related air pollution and noise[END_REF][START_REF] Spicer | Chemical composition of exhaust from aircraft turbine engines[END_REF]. Different types of volatile PAHs were identified using a sampler placed outside in a residential area very close to an airport. It is interesting to note that the concentration of the particulate phase measured near homes was similar to that at the end of runways [START_REF] Jung | Personal, indoor, and outdoor exposure to VOCs in the immediate vicinity of a local airport[END_REF].

It has been suggested that aviation can cause largescale increases (.30%) of black carbon particles in the upper troposphere and lowermost stratosphere of regions highly frequented by aircraft [START_REF] Hendriks | Simulating the global atmospheric black carbon cycle: a revisit to the contribution of aircraft emissions[END_REF]. These particles are then disseminated in the atmosphere. UFPs represented 15-18% of the particulate concentration in an airport flight path atmosphere (Taiwan International Airport, Taipei, Taiwan) [START_REF] Lai | Characteristics of nano-/ultrafine particle-bound PAHs in ambient air at an international airport[END_REF] and this rate varied depending on the engine, the type of fuel and climate conditions [START_REF] Kobayashi | Increased formaldehyde in jet engine exhaust with changes to JP-8, lower temperature, and lower humidity irritates eyes and respiratory tract[END_REF]. Some studies outline that soot particles could have adverse health effects, especially on the respiratory tract, because of their tiny size [START_REF] Highwood | When smoke gets in our eyes: the multiple impacts of atmospheric black carbon on climate, air quality and health[END_REF]. Experimental studies have demonstrated that the elementary particle size in airports or close to aircrafts was between 23 and 36 nm [START_REF] Barone | Inertial deposition of nanoparticle chain aggregates: theory and comparison with impactor data for ultrafine atmospheric aerosols[END_REF][START_REF] Smekens | Characterisation of soot emitted by domestic heating, aircraft and cars using diesel or biodiesel[END_REF]. Recently, around Los Angeles International Airport (LAX; Los Angeles, CA, USA), aircraft UFP emissions with a median size of ,11 nm were observed [START_REF] Hu | Aircraft emission impact neighborhood adjacent to a general aviation airport in southern California[END_REF]. This is consistent with the observation at LAX that there was a bimodal distribution of particle size showing peaks at ,12 and 80-90 nm diameter [START_REF] Westerdahl | The Los Angeles international airport as a source of ultrafine particles and other pollutant to nearby communities[END_REF].

An analysis of atmospheric health impacted around O'Hare International Airport (Chicago, IL, USA) indicated that the concentrations of the aforementioned pollutants, although high, especially downwind, were similar to those from road traffic emissions in urbanised areas [16].

It is therefore difficult to focus specifically on air traffic pollution because the rates of pollutants are the same as those found in highly urbanised areas [START_REF] Tesseraux | Risk factors of jet fuel combustion products[END_REF].

AIRPORT VICINITY

In fact, several studies, especially in Europe and North America, have been conducted since the 1990s to assess the health impact on people living close to airports, as well as air traffic risks such as noise and air pollution. However, there is a lack of data regarding relationships between airport atmospheric pollutants and respiratory disorders.

In the Netherlands, the health authorities are interested in the topic, and many studies have been conducted at the Schiphol hub airport (Amsterdam, the Netherlands) and in its vicinity. It has been determined that people living near Schiphol complained about respiratory problems [START_REF] Staatsen | breath, wheezing onset and decreased lung function[END_REF] comprising coughing, an increase in the perception of shortness of breath and wheezing onset associated with a decreased lung function. An increase in the intermittent use of bronchodilators was noted for people previously suffering from chronic respiratory diseases; they decompensated more often [4]. These functional signs were most frequently identified near the hub and decreased with the distance from the airport [START_REF] Staatsen | breath, wheezing onset and decreased lung function[END_REF]. This notion confirmed results of a study conducted in the USA. A significantly larger number of hospitalisations due to acute respiratory symptoms was highlighted for people living within 8 km of major airports like LaGuardia Airport (New York, NY, USA) or Greater Rochester International Airport (Rochester, NY) compared with those living beyond 8 km [19]. The intensity of respiratory symptoms evolved according to the concentration of air pollution.

The Dutch Working Group focused on the issue of airport respiratory health impact on vicinity and concluded that currently there was no direct association between airport air pollution and other respiratory problems other than those encountered in areas of high urban pollution [4].

The various studies are very descriptive and include many confounding factors, so we cannot compare or draw conclusions with certainty. To further support the hypothesis of an impact on respiratory functions directly related to airportspecific air pollution, work should be carried out with the target populations that are most at risk, such as employees who work in hub airports.

AIRPORT WORKERS: THE POPULATION MOST EXPOSED

In 1999, the effect of occupational exposure to aircraft fuel and jet exhaust on pulmonary function and respiratory symptoms in Birmingham International Airport (Birmingham, UK) workers was reported. This work suggested that there was a link between high occupational exposure to aviation fuel or jet exhaust and excess respiratory tract symptoms, consistent with the presence of a respiratory irritant. It was a cross-sectional survey using a questionnaire filled out by the participants themselves. Respiratory and immunological function assessments and an exhaled carbon monoxide measurement were conducted with male full-time airport workers classified into three groups according to their exposure level [START_REF] Tunnicliffe | Pulmonary function and respiratory symptoms in a population of airport workers[END_REF].

Although neither spirometry nor skin tests demonstrated a difference between groups, it appeared that high exposure is significantly associated with a runny nose and a cough with phlegm. The odds ratios were 2.9 and 3.5, respectively (p,0.05), after adjustment for age, smoking and seasonal rhinitis. Furthermore, no obvious link was demonstrated between the presence of shortness of breath or wheeze and high exposure (table 1). Exhaled carbon monoxide, adjusted for smoking, was similar for all groups. However, these results need to be confirmed by further studies because they were limited by a lack of quantitative data on occupational exposure compared with unexposed workers and the small number of subjects involved [START_REF] Tunnicliffe | Pulmonary function and respiratory symptoms in a population of airport workers[END_REF].

In 2003, American researchers studied the prevalence of respiratory symptoms in flight attendants because the indoor environment in occupational settings had generated considerable concerns in the USA. An association between respiratory disorders after chronic and high exposure to aircraft air quality was suggested (long-haul rotations supported). It was stated that the aircraft cabin air supplies are first cycled through an aircraft engine. Flight attendants were asked standardised questions on respiratory health by telephone at three different international airports (Miami International Airport, Miami, FL, USA; Seattle-Tacoma International Airport, Seattle, WA, USA; and Detroit Metro Airport, Detroit, MI, USA) concerning their respiratory health. Teachers operating in the same areas constituted the control group (matched for sex and age, but with little air travel). Interviewers used a standardised questionnaire [21]. Flight attendants reported significantly more chest illnesses than teachers (33% and 19.3%, respectively), although doctordiagnosed asthma was less common in flight attendants. The authors concluded that flight attendants had a higher respiratory disorder rate than other American female workers. The lack of asthma diagnosis might be explained by a work selection bias (inability to fly if asthma is pathology proven). The occupational origin of these symptoms was highlighted because the air cabin environment represented a unique occupational setting [21].

Pressure, humidity or many people in a small area (increased risk of infections) are specific to aircraft cabins, and the authors also outlined some contaminants that are more concentrated than in other environments: ozone, degradation products of the combustion of engine oils or hydraulic fluids. It remains difficult to go beyond hypothesis as clinical data were not associated with workplace metrology results or an objectification of medical symptoms. The link between cabin air potentially contaminated by particles of secondary emissions from jet-fuel combustion and respiratory problems could not be assessed here; there were too many missing data and too much bias [21].

In the early 2000s, a working group, appointed by the Committee on Toxicology of the US National Research Council, studied the issue of health effects of aviation fuel in the US Air Force (fig. 2). Several internal studies at the US Air Force were conducted with in the course of their work.

The working group reported the results of two studies concerning the breathing impact from Jet Propellant (JP)-8 (the main fuel used by military aviation): one by self questionnaire on respiratory symptoms of three groups of soldiers with different exposure levels; and the other based on a cohort followed by identifying the number of Air Force medical consultations for respiratory disorders compared with a control group that was not exposed. No significant difference could be demonstrated between these groups in both cases.

However, further investigations were recommended by conducting more rigorous studies taking into account the fuel in FIGURE 2. High-idling aircraft engine. Data are presented as %, unless otherwise stated. Data from [START_REF] Tunnicliffe | Pulmonary function and respiratory symptoms in a population of airport workers[END_REF].

its various states, to better characterise the workplace atmosphere and, therefore, collect clinical information and find an association with metrological data. In vitro studies on mice suggested that JP-8 aerosol is more toxic to the respiratory tract than JP-8 in vapour form [START_REF]Subcommittee on Jet-Propulsion Fuel 8[END_REF].

To assess the potential exposure to PAHs emitted in airports, either due to the combustion of JP-8 or from other sources (road vehicles, maintenance operations, etc.), an American team attempted to characterise the ambient aerosol in various US Air Force workplaces (fig. 3).

Different kinds of measurements were used: 1) PAH concentration rendered in real-time monitoring (photoelectric aerosol sensor); and 2) low-volume air samplers for chemical analysis (gas chromatography (GC)/mass spectrometry (MS)). These monitors were used at the same time. The results were concordant on a significant increase in the concentration of PAHs in the different workplaces studied. The PAH rate was up to 25 times the content usually found in the ambient air. GC/MS clarified that PAHs were more present in a volatile phase with naphthalene and alkyl-substituted naphthalenes. Particulatephase PAHs were below the method detection limits. It is interesting to note that GC/MS revealed specific PAHs in workplaces with a major diesel exhaust particle (DEP) exposure; these compounds were not detected close to jet exhaust.

They concluded that occupational exposure to PAHs is real but it is difficult to associate a potential impact on health with this exposure. In fact, the rate measured in the break room or in the hangar, in the absence of major maintenance activities on rolling aircraft, is similar to that which was measured with the same type of monitor in the ambient air of major urban residential areas. The maximum concentration ''on the tarmac'' during an engine test (four reactors) was twice as large as the rate measured in a ''smoking office''. The emphasis was on high risk of acute exposure over short periods during certain activities (concentration peaks of PAHs) (table 2) [START_REF] Childers | Real-time and integrated measurement of potential human exposure to particlebound polycyclic aromatic hydrocarbons (PAHs) from aircraft exhaust[END_REF].

These relatively low levels of exposure to PAHs from the combustion of aviation fuels were mainly related to the presence of naphthalene, alkyl-substituted naphthalenes and, in smaller quantities, fluoranthene, pyrene and benzo[a]pyrene. Other studies confirm these data [START_REF] Iavicoli | Determination of airborne polycyclic aromatic hydrocarbons at an airport by gas chromatography-mass spectrometry and evaluation of occupational exposure[END_REF].

An assessment of the occupational exposure of the airport ground staff at the hub airport Charles de Gaulle (Paris, France) was carried out by occupational physicians. Atmospheric measurements were made from integrated-air samples and with individual portable tools (carbon monoxide analyser and aromatic hydrocarbons pump), specifically during the towing of the aircraft, near the towing personnel. The hydrocarbon emissions were higher under idle or low-load conditions compared with high-load conditions. It was during this activity that employees were most exposed. These results in commercial aircraft confirmed the report by CHILDERS et al. [START_REF] Childers | Real-time and integrated measurement of potential human exposure to particlebound polycyclic aromatic hydrocarbons (PAHs) from aircraft exhaust[END_REF] that the rate of PAHs is very low, except at certain times when peaks were detected. Nitrogen oxides measured in a fixed position were below the occupational exposure limits. Carbon monoxide was below the detection limit of the device and stationary particles measured were 100 times lower than the regulatory values. The author states that all these data were conditional because the weather was not taken into account and, therefore, they only reflected the airport's ambient environment at one moment [START_REF] Donne | Prevention of airport hazards on tarmac: 1. Noise, 2. Chemical and particles pollution[END_REF].

To better characterise the occupational exposure to PAHs, an Italian team decided to assess the association between atmospheric metrology (measuring levels of 23 kinds of PAHs including 16 considered most dangerous by the US Environmental Protection Agency) and a urinary PAH indicator (1-hydroxypyrene) in the exposed population, and to assess micronucleus and comet assays in exfoliated buccal cells. PAHs were found in greater numbers on the Fiumicino-Leonardo da Vinci (Rome, Italy) apron with predominantly methylnaphthalene and acenaphthylene compared with other areas (buildings and departure terminal). The authors associated PAHs with the incomplete combustion of jet exhaust. The analysis of the rate of 1-hydroxypyrene in urine, collected at the end of the fifth day of the work week showed no significant difference between the exposed workers and the control group. Conversely, although the micronucleus test was not significant, the comet assay (confirmed by secondary analyses after culture) showed the presence of oxidative stressrelated damage to DNA. CAVALLO et al. [START_REF] Cavallo | Occupational exposure in airport personnel: characterization and evaluation of genotoxic and oxidative effects[END_REF] concluded that the comet assay on buccal cells was a good indicator of genotoxic and oxidative impact after chronic exposure to low doses of PAH. However, this is not clear; there are many biases in this study, with a lack of precision on the exposure time and a neglect of numerous confounding factors [START_REF] Cavallo | Occupational exposure in airport personnel: characterization and evaluation of genotoxic and oxidative effects[END_REF].

However, alkanes emitted by aircraft engines may be a specific aircraft emission indicator for occupationally exposed persons, as their concentration is higher than in DEPs [START_REF] Zeiger | The First International Conference on the Environmental Health and Safety of Jet Fuel[END_REF], and the impact of other airport respiratory toxic products must not be neglected. Occupational physicians should consider the global exposure.

Paints may contain VOCs that are implicated in the exacerbation of pre-existing respiratory diseases such as asthma [START_REF] Lapuma | The impact of recirculating industrial air on aircraft painting operations[END_REF]. In addition, chromium found in spray paints used in the aviation industry seems to be deposited in the airway and alveolar tract [START_REF] Sabty-Daily | Size distribution and speciation of chromium in paint spray aerosol at an aerospace facility[END_REF]. Aircraft maintenance is often performed in closed, ventilated areas (hangars). These operations are very different and may require the use of paints, adhesives, oils or welding. It has been FIGURE 3. Traffic vehicles on runways.

shown that this last activity is a source of not only organic UFPs but also metal UFPs such as aluminium, iron or zinc [START_REF] Zimmer | Investigation of the aerosols produced by a high-speed, hand-held grinder using various substrates[END_REF].

DISCUSSION

The health impact of airport occupational exposure cannot be fully considered while the risks have not been clearly identified. No study has demonstrated a significant relationship between specific exposure to jet exhaust particles and respiratory symptoms because of many confounding factors (tobacco, DEPs, etc.), insufficient sample size and a lack of quantitative data; nobody has been able to correlate jet exhaust particles with respiratory symptoms in fact. Many studies on animals and humans (in vitro and in vivo) regard nanoparticle toxicity but this research focuses on workers in nanotechnology industries who are exposed to uniquely engineered nanomaterials. Therefore, it is easier to include this kind of person in a epidemiological study with few confounding factors. However, this field of study should also include nanoparticles generated by pollution as well as manmade nanoparticles, UFPs remain a background interest despite a larger number of people being exposed. This may be because natural UFPs were considered common in the atmosphere, but with the development of industry and travel traffic, people are to significant levels.

Since the air in dense urban areas does not differ from that of an airport, we must take into account atmospheric pollution in its entirety. Traffic at an airport is not only limited to aircraft runways; it also has a lot of roads for cars and cargo trucks. Furthermore, it covers a larger number of activities on a smaller geographic scale. As in urban areas, where pollution from DEPs has been proven, it is important to remember that the International Agency for Research on Cancer recently classified diesel engine exhaust as carcinogenic to humans (Group 1). This is based on sufficient evidence that exposure to DEPs is associated with an increased risk of lung cancer. The exhaust of gasoline engines is also suspected as carcinogenic (Group 2B) [START_REF]Diesel and gasoline engine exhausts and some nitroarenes[END_REF]. Therefore, toxicity due to other types of fuels must also be discussed.

Since the impact of respiratory UFPs derived from the combustion of diesel is more and more accurate, we can begin to question the existence of these particles in an airport, outdoors and indoors. We can also better characterise these particles since the majority of road vehicles use diesel, especially in France, and aeroplanes consume kerosene. A team of researchers in Marseilles has recently demonstrated in vitro that these UFPs can influence cytokine production and so impact inflammatory processes in humans [START_REF] Ferry | Jet exhaust particles alter human dendritic cell maturation[END_REF]. For an improved approach to occupational exposure on airport aprons, the balance of each of these types of pollution should also be determined (diesel versus kerosene).

It would also be interesting to see if there is an association between ambient air metrology and objective respiratory health indicators (biometrology, functional medical exams, etc.).

Depending on the impact that can be measured, key prevention and corrective methods may be considered, on a human scale (collective and individual) as well as an industrial scale.

Until a specific marker of incomplete combustion of kerosene is clearly identified, it is necessary to continue the process of RTM: real time measurements; IAS: integrated air sampling associated with chemical analysis; LD: limit of detection; AGE: aerospace ground environment. # : present in the greatest quantities. Data from [START_REF] Childers | Real-time and integrated measurement of potential human exposure to particlebound polycyclic aromatic hydrocarbons (PAHs) from aircraft exhaust[END_REF].

measuring the concentration in ambient air on the workforce, and on a larger scale, for public health. The specificity of alkanes has been discussed and should continue to be studied before being used as a marker. The difference in size between UFPs from jet exhausts and those from DEPs must be taken into consideration.

It is important to continue studies in order to characterise these pollutant substances because data gathered from other sectors attributes toxicity to the nanometric size of these particles. We know these particles have different physical and chemical properties relating to their size.

Given the enormous increase in air traffic and the controversies it has generated in recent years, airport structures are changing, not only in terms of their architecture. The use of fuel in aircrafts and road vehicles is also being redesigned (electric vehicles, organic jet fuel, etc.). There are still insufficient data and new factors must also be considered.

The monitoring of the Swiss Study on Air Pollution and Lung Disease in Adults cohort reported that an improvement in air quality due to behavioural changes had a positive impact on respiratory health [START_REF] Downs | Reduced exposure to PM10 and attenuated age-related decline in lung function[END_REF].

CONCLUSION

It is clear that airport activities are a source of air pollution. However, jet exhausts are not the only cause of this pollution. The wide range of operations on an airport base increases occupational exposures. Only a few studies have established a specific link between exposure to pollution in an airport work environment and respiratory problems (table 3). Moreover, the correlation is weak because there is a lack of power, a lack of precision over the types of jobs affected and the presence of confounding factors.

Additional studies, using more rigour and more biomonitoring in association with occupational aerosol measurements, are needed to support this hypothesis.

STATEMENT OF INTEREST

FIGURE 1 .

 1 FIGURE 1. Jet exhausts in the sky.

  working time on the airport apron, some in reasonable proximity to aircraft and some within the terminal buildings

TABLE 1

 1 Occupational exposure of Birmingham International Airport (Birmingham, UK) workers and respiratory disorders

	Crude prevalence of respiratory problems		Runny nose Cough with Shortness of Wheezing	phlegm breath
	Median time spent	in aircraft taxiing	area h?day -1
	Comments			Considerable proportion of their working day
	Subjects n			53
	Workers			Baggage handlers	Airport hands	Marshallers	Operational engineers	Fitters	Engineering technicians
	Exposure	group		High

in close proximity to in-service aircraft

TABLE 2

 2 Airport occupational exposure to polycyclic aromatic hydrocarbons (PAHs)

	Workplace	Monitor	Results
	Background in a break room close to mainte-	RTM	0.97-40 ng?m -3
	nance hangar (no known emission sources		Negligible
	near the room)		The monitors all agreed qualitatively
	Downwind measurements during four engine	RTM	Maximum output of the monitor: 4000 ng?m -3 (high-idle engine test)
	run-up test on flight tarmac	IAS	PAH vapour phase dominated by naphthalene, and alkyl-substituted naphthalenes #
			PAH particulate phase ,LD except benzo[a]pyrene and pyrene
	Maintenance hangar, first step: minimal flight and	RTM	Negligible (similar to the break room)
	ground-support activity (maintenance hangar	IAS	PAH vapour phase dominated by naphthalene and alkyl-substituted naphthalenes #
	background)		PAH particulate phase ,LD
	Maintenance hangar, second step: doors opened;	RTM	PAHs were 10 times higher than in the break room, eight times higher than those
	aircraft engine exhaust was directed toward the		recorded earlier in the maintenance hangar (first step), approximately equal to the
	door of the maintenance hangar		average indoor PAH concentrations in residences in a major city
		IAS	Vapour PAHs were two times greater than in the maintenance hangar (first step)
			PAH particulate phase ,LD
	Downwind from aerospace ground equipment	RTM	The response reached a maximum at the AGE units start and shut off
			20 times greater than the background levels, 10 times higher than in the maintenance
			hangar (first step), 3.5 times higher than during the four-engine run-up test
		IAS	New vapour PAHs were detected: methyl substituted phenanthrenes, anthracenes,
			fluoranthene and pyrene
			PAHs particulate phase ,LD

Conflict of interest information can be found alongside the online version of the article at err.ersjournals.com


André Alves Dias

Dominique Raze

Cristiana Soares De Lima

Maria Angela De Melo Marques

Hervé Drobecq

Anne-Sophie Debrie

Michelle Lopes

Franck Biet

Maria Cristina

Vidal Pessolani
email: cpessola@ioc.fiocruz.br

Michelle Lopes Ribeiro-Guimarães

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

online | memorias.ioc.fiocruz.br Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 107(Suppl. I): [174][175][176][177][178][179][180][181][182]2012 Despite decades of investigations, tuberculosis and leprosy caused by Mycobacterium tuberculosis and Mycobacterium leprae, respectively, still represent major threats to human health. If M. leprae is a major concern in developing countries with an incidence of about 230.000 new cases per year (WHO 2010a), M. tuberculosis remains the world's leading cause of death due to a single infectious agent with 1.5 million deaths annually (WHO 2010b). Although these two bacterial species display different tropisms for human tissues, they both trigger infections characterized by a systemic dissemination step which requires specific interactions of the pathogen with host cell [START_REF] Pessolani | Systemic dissemination in tuberculosis and leprosy: do mycobacterial adhesins play a role?[END_REF]. The mechanisms responsible for the systemic mycobacterial dissemination remain poorly understood and their deciphering constitutes a major challenge since it may lead to the development of new prophylactic and/or therapeutic strategies. To better understand the molecular interplay between mycobacteria and host cells, we have initiated investigations dealing with the identification and characteriza-tion of mycobacterial adhesins that interact with human host cells. These studies led us to identify a 22-kDa surface-exposed heparin-binding haemagglutinin adhesin (HBHA) which is involved in the interaction of M. tuberculosis and M. leprae with epithelial cells, but not with professional phagocytes [START_REF] Menozzi | Mycobacterium tuberculosis heparin-binding haemagglutinin adhesin (HBHA) triggers receptormediated transcytosis without altering the integrity of tight junctions[END_REF][START_REF] Lima | Heparin-binding hemagglutinin (HBHA) of Mycobacterium leprae is expressed during infection and enhances bacterial adherence to epithelial cells[END_REF]). In addition, HBHA which binds heparan sulphatecontaining receptors through its C-terminal lysine-rich domain [START_REF] Delogu | Functional domains present in the mycobacterial hemagglutinin, HBHA[END_REF][START_REF] Pethe | Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin[END_REF] has been shown to be required for extrapulmonary dissemination [START_REF] Pethe | The heparin-binding haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination[END_REF][START_REF] Mueller-Ortiz | Decreased infectivity despite unaltered C3 binding by a DeltahbhA mutant of Mycobacterium tuberculosis[END_REF]. Recently, HBHA was shown to trigger receptor-mediated transcytosis, suggesting that mycobacteria may disseminate via a direct passage through an epithelial barrier [START_REF] Menozzi | Mycobacterium tuberculosis heparin-binding haemagglutinin adhesin (HBHA) triggers receptormediated transcytosis without altering the integrity of tight junctions[END_REF]. Besides HBHA, the histone-like protein (Hlp) has also been implicated in the attachment of pathogenic mycobacteria to host cells. Mycobacterial Hlp, a positively-charged, surface-exposed molecule with roughly twice the size of other bacterial Hlps, is a highly conserved protein shared by all mycobacterial species [START_REF] Lefrançois | Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/ histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease[END_REF]. This protein was initially described as a laminin-binding protein (LBP) involved in M. leprae-Schwann cell (SC) interaction [START_REF] Shimoji | A 21-kDa surface protein of Mycobacterium leprae binds peripheral nerve laminin-2 and mediates Schwann cell invasion[END_REF][START_REF] Marques | Bacterial and host-derived cationic proteins bind alpha2-laminins and enhance Mycobacterium leprae attachment to human Schwann cells[END_REF]. More recently LBP/Hlp has been shown to also play a major role in mediating the adhesion of mycobacteria to epithelial respiratory cells by interacting with proteoglycan-containing receptors such as heparan sulphate and hyaluronic acid (HA) [START_REF] Aoki | Extracellular mycobacterial DNA-binding protein 1 participates in mycobacterium-lung epithelial cell interaction through hyaluronic acid[END_REF][START_REF] Lefrançois | Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/ histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease[END_REF].

When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a wellcharacterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/ Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

Because collagenous proteins are the major constituents of the extracellular matrices of epithelial cells and may serve as receptors for bacterial adherence [START_REF] Kreis | Guidebook to the extracellular matrix and adhesion proteins[END_REF], we have conducted experiments to know whether mycobacteria may directly interact with collagen. In the present study, we show that Mycobacterium bovis BCG grown in the presence of exogenous collagen I forms clusters. In addition, cytoadherence assays have revealed that the interaction of M. bovis BCG with pneumocytes and macrophages is increased in the presence of exogenous collagen or following a pretreatment of the mycobacteria with collagen. These observations indicated that M. bovis BCG is able to recruit collagen, suggesting the presence of a surface-exposed collagen-binding receptor. This receptor has been purified by affinity chromatography and demonstrated to be the LBP/Hlp protein. Finally, we mapped the collagen-binding activity of LBP/ Hlp within the C-terminal domain of the protein.

MATERIALS AND METHODS

Mycobacterial strains and growth conditions -M. bovis BCG (strain 1173P2, World Health Organization, Stockholm, Sweden) was grown at 37ºC in tissue culture grade Roux flasks (Nunc) using Sauton medium supplemented or not with type 1 calf skin collagen (Collagen S, Roche, Mannheim, Germany) at the concentration of 25 µg/mL. Mycobacterium smegmatis wild-type (wt) mc 2 155 and the mutant for the hlp gene (Δhlp) were kindly provided by Thomas Dick of Singapore University, Singapore. The mutant was generated as previously described by [START_REF] Lee | Upregulation of a histonelike protein in dormant Mycobacterium smegmatis[END_REF]. Both of the strains were grown in Middlebrook 7H9 broth (Difco, Detroit, MI, USA) supplemented with 10% albumin, dextrose and NaCl (ADC) and 0.05% Tween-80 and 0.5% glycerol under agitation. Mycobacteria were acid-fast stained by the Kinyoun method and visualised under light microscope.

Mycobacterial adherence assays -Exponentiallygrowing M. bovis BCG (OD 600 of 0.4) were collected by low-speed centrifugation (6.500 g for 10 min at 4ºC) and carefully resuspended in 50 mM Tris-HCl (pH 7.3) containing 150 mM NaCl [Tris buffered saline (TBS)]. The mycobacterial cytoadherence was then assayed as described previously [START_REF] Menozzi | Identification of a heparin-binding hemagglutinin present in mycobacteria[END_REF]) using A549 cells (human type II pneumocytes; ATCC, CCL 185) or U937 cells (human macrophages; ATCC, CCL 1593) grown in 24-well tissue culture trays. The adherence assays were performed at a multiplicity of infection of 10 in the absence or the presence of increasing concentrations of type 1 collagen. Following 2 h of incubation at 37ºC, the cells were washed three times with TBS, lysed by adding 1 mL distilled water containing 0.05% (wt/vol) sodium deoxycholate and serial dilutions were plated onto 7H11 medium for colony forming units (CFUs) counting. Cytoadherence is expressed as the percentage of CFUs present in the inoculum that remain associated with target cells after the washing step. To investigate the effect of trypsin or collagen pretreatment on the M. bovis BCG cytoadherence, the bacteria were incubated for 30 min. at 37ºC in TBS supplemented with porcine pancreas trypsin (Sigma) or collagen I, respectively. After three washes with TBS, the bacilli were passed five times through a 28-gauge needle to unravel possible clumps and finally submitted to the adherence assay as described above.

Affinity chromatography on collagen-Sepharose -Collagen I was covalently linked to CNBr-activated Sepharose 4B (Amersham) according to the manufacturer's recommendations. Briefly, 5 mL of gel swollen in distilled water was washed with 20 mL 1 mM HCl followed by 200 mL phosphate buffered saline (PBS) (10 mM phosphate buffer pH 7.2, 0.15 M NaCl). The gel was then resuspended in 25 mL PBS containing collagen I at the concentration of 1 mg/mL and incubated overnight at 4ºC under gentle agitation. The remaining active groups were blocked by washing the gel with 200 mL 100 mM Tris-HCl pH 8.0. The collagen coupling efficiency ranged between 95-99%. Fractions containing soluble extracts of M. bovis BCG were prepared as described previously [START_REF] Menozzi | Identification of a heparin-binding hemagglutinin present in mycobacteria[END_REF]) and chromatographed at a flow rate of 1.5 mL/min over 5 mL of collagen-Sepharose matrix packed in a glass column (1-cm diameter) and equilibrated with PBS. At the end of the sample loading step, the gel was washed with 100 mL PBS and the bound material was eluted by a 0-1 M NaCl linear gradient in 100 mL PBS. Fractions of 1 mL were collected and their protein concentrations were determined by using the bicinchoninic acid method (Pierce) and bovine serum albumin (BSA) as a standard.

Protein identification -The protein band corresponding to the M. bovis BCG collagen-binding protein was excised from the Coomassie-stained polyacrylamide gel, washed twice with 50% acetonitrile prepared in 20 mM ammonium hydrogenocarbonate and finally digested overnight within the gel fragment using 50 ng trypsin (Promega, Madison, WI, USA). The resulting peptides were eluted, desalted with a ZIPTIP C18 column (Millipore, Billerica, MA) and spotted on a Maldi plate with 0.5 µL of freshly dissolved α-cyano-4-hydroxycinnaminic acid at 5 mg/mL in 50% acetonitrile and 0.1% trifluoroacetic acid. After drying, the spots were washed with 3 µL of 20 mM diammonium citrate pH 4.5 and mass spectrometry analyses were performed by using a matrix-assisted laser desorption ionization/time-of-flight Voyager-DE-STR (Applied Biosystems, Palo Alto, CA). Peptides were analyzed by using the following setting parameters: positive and reflector modes, acceleration voltage of 20 kV, grid voltage of 61%, 90 ns of delayed extraction time and low mass gate 500 Da. The spectra were calibrated externally by using the [M+H + ] monoisotopic ions of peptides resulting from trypsin-digested lyzozyme. Database search based on the peptide masses observed was performed against the NCBInr database using the MS-FIT algorithm (Protein Prospector;128.40.158.151/ mshome3.4.htm) or the ProFound algorithm (prowl.rockefeller.edu/profound_bin/WebProFound.exe).

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis

-SDS-PAGE was performed using a 12-15% gel and proteins were stained with Coomassie brilliant blue R-250 (ICN) or silver (Sigma). Alternately, proteins were trans-ferred onto nitrocellulose membranes (Protran BA85, Schleicher & Schuell) as described. Immobilized proteins were probed with the 5G9 anti-rLBP/Hlp monoclonal antibody (1:1000) [START_REF] Marques | Bacterial and host-derived cationic proteins bind alpha2-laminins and enhance Mycobacterium leprae attachment to human Schwann cells[END_REF]. The membranes were then washed three times with TBS/T and incubated with a goat anti-mouse alkaline phosphateconjugated antibody. The substrates nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were used for colour development.

Scanning electron microscopy (SEM) -M. bovis BCG grown in Sauton medium to mid log phase in the absence or the presence of 25 µg/mL collagen I were resuspended in PBS (OD 600 of ~1.0). The bacteria were then fixed for 5 h at room temperature (RT) in a 1.25% (v/v) glutaraldehyde solution prepared in 100 mM sodium cacodylate buffer (pH 7.0). A similar fixation step was applied to a collagen I solution at 50 µg/mL prepared in PBS. Next, the samples were filtered through 25 mm diameter/0.2 µm porosity Anodisc (Whatman) and the filters were rinsed five times for 10 min in 25 mL cacodylate buffer. Post-fixation was performed for 3 h in 1% OsO 4 solution prepared in cacodylate buffer, followed by five washes in ultrapure water. The samples underwent progressive dehydration by successive soaking in 50%, 70%, 95% and 100% ethanol. Soaking in isopentyl acetate was performed before critical point drying in CO 2 , using an EMSDCOPE CPD 750 apparatus. The filters were then attached to large SEM stubs and coated with gold/palladium by cathodic spreading in a Polaron E5100 coater. Sample observation and microphotographs were done in a JEOL JSM35CF scanning electron microscope, operating at a voltage of 10 kV.

Preparation of recombinant full-length LBP/Hlp

and truncated forms -The recombinant M. bovis BCG LBP/Hlp protein was obtained using the same procedure previously described for cloning and expression of the Mycobacterium avium subsp. paratuberculosis LBP/Hlp-encoding gene [START_REF] Lefrançois | Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/ histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease[END_REF]). The M. bovis LBP/Hlp-encoding gene (GenBank accession GQ259334) was amplified by polymerase chain reaction from chromosomal DNA of M. bovis BCG strain 1173P2 using the Pfu DNA polymerase (Promega) and two synthetic oligonucleotides (Sigma) with the following sequences: 5'TATACATATGCACCACCACCA CCACCACATGAACAAAGCAGAGCTCATTGACG -3' (BCG-LBP/Hlp S) and 5'-TATAGCGGCCGCCT-ATTTGCGACCCCGCCGAGCGG-3' (BCG-LBP/Hlp AS), containing an NdeI and NotI sites, respectively. The oligonucleotide BCG-LBP/Hlp S sequence was designed to produce a hybrid protein carrying a His-tag at the Nterminal position used for affinity purification of the recombinant protein. Recombinant full-length M. leprae LBP/Hlp (rLBP/Hlp) and truncated forms of the protein corresponding to the N-terminal (residues 1-110; rLBP/ Hlp-N) and the C-terminal (residues 111-200; rLBP/ Hlp-C) were obtained as previously described [START_REF] Marques | Bacterial and host-derived cationic proteins bind alpha2-laminins and enhance Mycobacterium leprae attachment to human Schwann cells[END_REF][START_REF] Lima | Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae[END_REF].

Binding assays in microplates of recombinant BCG LBP/Hlp to collagen I -To investigate the capacity of M. bovis BCG rLBP/Hlp to bind to collagen, 1.0 μg/mL of collagen I (Sigma, St. Louis, MO) in 50 μL 0.1 M carbonate-bicarbonate buffer (pH 9.6) was used to coat the wells of a polystyrene microplate (Corning, New York, NY). The microtitre plate was incubated overnight at 4ºC. The wells were then washed with PBS and blocked for 2 h with 200 μL PBS-3% BSA at RT. After washing with PBS/0.05% Tween 20 (PBS/T), 50 μL of increasing concentrations of M. bovis BCG rLBP/Hlp were added to the wells and incubated at 37ºC for 2 h. The wells were then rinsed with PBS/T and incubated with the anti-LBP/Hlp monoclonal antibody 5G9 (1:500) for 1 h at 37ºC. After washing with PBS/T, rabbit anti-mouse IgG peroxidase conjugate (Sigma; 1:1000) was added and incubated for an additional 50 min at 37ºC. Peroxidase activity was revealed with hydrogen peroxide and o-phenylenediamine (OPD). The reaction was stopped with HCl and read at 490 nm with a TitertekPlus microplate reader (ICN Biomedicals Inc, Costa Mesa, CA). Control wells coated with BSA were included in all binding assays.

Mapping the collagen-binding site of M. leprae LBP/

Hlp -To monitor the binding of different types of soluble collagens to M. leprae rLBP/Hlp or truncated forms, 5 µg/mL of each protein in 0.1 M carbonate buffer pH 9.6 (50 µL) were used to coat the wells of polystyrene microplates (Corning, New York, NY). Plates were incubated overnight at 4ºC. The wells were then washed with PBS and blocked for 2 h at RT with 200 µL PBS containing 2% BSA. Upon washing with PBS/T, 50 µL of increasing concentrations of biotinylated collagens I, III, IV or VI (Sigma; 0-80 µg/mL) were added to the wells and incubation was performed at RT for 2 h. The wells were rinsed with PBS/T and incubated with streptavidin-peroxidase (Pierce, Rockford, IL) at 0.5 µg/mL. Peroxidase activity was revealed with hydrogen peroxide and OPD. The reaction was stopped with HCl and read at 490 nm in a TitertekPlus microplate reader. In control wells, collagen was omitted and specific collagen-binding activity was determined by subtracting the absorbency resulting from non-specific binding detected in the control wells.

RESULTS

Collagen-induced agglutination of M. bovis BCG

-To investigate the interaction of M. bovis BCG with collagen, we first cultivated the mycobacteria in Sauton medium in the absence or the presence of collagen I at the concentration of 25 µg/mL. In the control culture, the mycobacteria formed a thin layer that stuck loosely to the bottom of the Roux flask and the cells were easily resuspended by repeated shakings of the flask. When collagen I was added to the culture medium, the bacterial layer was more firmly bound to the flask surface and required the use of a cell scraper to completely detach the cells. In addition, once in suspension the mycobacteria were not evenly dispersed, but most of them remained agglutinated in small clumps. To examine the structure of these clumps, mycobacteria were observed by SEM. As shown in Fig. 1A and Supplementary data, M. bovis BCG cells grown in the absence of collagen failed to form aggregates and most of the bacteria appeared isolated on the filter membrane. In contrast, large bacterial aggregates were observed using M. bovis BCG from cultures performed in the presence of collagen I (Fig. 1B, Supplementary data). An amorphous and fibrillar material in contact with the bacteria was also observed within these aggregates. Because this material could represent collagen fibres cross-linked by glutaraldehyde during the sample preparation, collagen I was fixed and observed in the same conditions. Collagen appeared as long and entangled filaments exhibiting various thicknesses (Supplementary data). Since this aspect was quite similar to that of the extracellular material detected within the mycobacterial clumps, it suggests that M. bovis BCG may interact directly with collagen fibres.

Exogenous collagen I increases the cytoadherence of M. bovis BCG -Because collagen is a predominant constituent of the extracellular matrices of epithelial cells [START_REF] Kreis | Guidebook to the extracellular matrix and adhesion proteins[END_REF], we assayed the cytoadherence of M. bovis BCG in the presence of increasing collagen I concentrations ranging from 0-100 µg/mL. These assays were performed using human type II A549 pneumocytes, but also U937 human macrophages since mycobacteria display a tropism for monocyte-derived phagocytic cells [START_REF] Stokes | Mycobacteriamacrophage interactions. Macrophage phenotype determines the nonopsonic binding of Mycobacterium tuberculosis to murine macrophages[END_REF][START_REF] Ernst | Macrophage receptors for Mycobacterium tuberculosis[END_REF]. As shown in Fig. 2, a dose-dependent effect was observed for both cell lines. Compared with the control, a ca. three-fold increase in adherence was observed with the A549 pneumocytes in the presence of 40 µg/mL collagen I. The use of higher collagen concentrations did not further increase the mycobacterial adherence, indicating a saturable mechanism. The effect of collagen I on the interaction of M. bovis BCG with U937 macrophages was also shown to be saturable, but it appeared more pronounced since a four-fold increase in adherence was observed in the presence of 20 µg/mL collagen I.

Pretreatment of M. bovis BCG with collagen I in-

creases its cytoadherence -In order to investigate the molecular mechanism leading to the increased cytoadherence of M. bovis BCG in the presence of exogenous collagen I, the bacilli were incubated in TBS containing 50 µg/mL collagen I prior to the cytoadherence assay. At the end of 30 min incubation at 37ºC, the mycobacteria were washed twice with TBS and then assayed for their capability to adhere to A549 pneumocytes and U937 macrophages. As shown in Fig. 3, such a collagen I pretreatment induced a ca. three-fold increase in the mycobacterial adherence onto both A549 pneumocytes and U937 macrophages. This observation suggests that M. bovis BCG may recruit exogenous collagen which may in turn favour the attachment of the bacilli to the eukaryotic cell surface. Such a recruitment was confirmed by the SDS-PAGE analysis of M. bovis BCG pretreated with collagen I followed by three washing steps with TBS. Indeed, the Coomassie blue staining of the gel revealed that collagen was present in the whole-cell lysate derived from mycobacteria pretreated with collagen (data not shown). This finding indicates that collagen I binds compounds probably associated with the M. bovis BCG surface. Therefore, these compounds could act as adhesins through their collagen-binding activity.

A proteinaceous M. bovis BCG compound is required for the collagen-dependent adherence -The recruitment of collagen by M. bovis BCG led us to characterize the mycobacterial surface compounds involved in this activ- ity. We first assayed the cytoadherence of mycobacteria pretreated for 30 min with porcine pancreas trypsin at the concentrations of 0.1 or 1 µg/mL. These conditions were chosen because neither modified the viability of M. bovis BCG, as observed by CFUs determination following the trypsin pretreatment (data not shown). Pretreatment of the bacilli with trypsin at 1 µg/mL did not significantly modify the mycobacterial interaction with U937 macrophages, suggesting that this interaction does not mainly involve proteinaceous adhesins (Supplementary data). In contrast, such a trypsin pretreatment induced a ca. 60% reduction of the M. bovis BCG attachment to the A549 pneumocytes. This finding is in agreement with the fact that HBHA, which is the major epithelial adhesin produced by both M. bovis BCG and M. tuberculosis, exhibits a surface-exposed heparin-binding adhesive domain highly sensitive to proteolytic degradation [START_REF] Pethe | Mycobacterial heparin-binding hemagglutinin and laminin-binding protein share antigenic methyllysines that confer resistance to proteolysis[END_REF][START_REF] Dupres | Nanoscale mapping and functional analysis of individual adhesins on living bacteria[END_REF]. When bacilli pretreated with 0.1 µg/mL trypsin were used in the cytoadherence assay run in the presence of 50 µg/mL exogenous collagen I, the increase in myco-bacterial adherence to both A549 pneumocytes and U937 macrophages was reduced by ca. 55% compared to untreated M. bovis BCG assayed in the presence of collagen (Supplementary data). This inhibitory effect of trypsin was confirmed by using M. bovis BCG pretreated with 1 µg/mL trypsin, as shown by the complete abolition of the collagen-induced stimulation of adherence onto A549 pneumocytes and U937 macrophages. This observation suggests that M. bovis BCG surface proteins are involved in the collagen-dependent cytoadherence.

LBP/Hlp binds immobilized collagen I -In order to isolate the M. bovis BCG compounds that bind collagen I, exponentially-growing mycobacteria were lyzed by sonication and centrifuged. Then, the centrifugation supernatant corresponding to the soluble material was collected and directly chromatographed onto a collagen I-Sepharose matrix equilibrated in PBS. Fractions corresponding to the eluted material were analyzed by SDS-PAGE and immunoblotting (Fig. 4A). Coomassie blue staining of the polyacrylamide gel revealed the presence of a protein exhibiting an apparent molecular weight of ca. 32 kDa. The gel band containing this protein was excised and submitted to trypsin digestion. Mass spectrometry analysis of the resulting peptides revealed peptide masses corresponding to the previously described mycobacterial LBP/Hlp. To confirm the collagen-binding capacity of BCG LBP/Hlp, the recombinant protein was successfully obtained (Supplementary data) and shown in microplate binding-assays to interact with collagen I (Fig. 4B). Finally, we cultivated M. smegmatis wt and Δlbp/hlp strains in 7H9 medium in the absence or presence of collagen I at the concentration of 25 µg/ mL. When cultures reached an OD 600nm of 1.8, bacteria were fixed, acid-fast stained and visualised under light microscope. Fig. 4C shows that, as expected, the mutant strain was unable to form large clumps, in contrast to the wt strain, indicating a decrease capacity to interact with collagen fibres when compared with the wt strain. These results confirmed the direct interaction between the mycobacterial LBP/Hlp and collagen.

Recombinant M. leprae LBP/Hlp binds collagen -M. leprae LBP/Hlp shares 84% sequence identity with the BCG homologue and it has been characterized as an important adhesin mediating bacterial interaction with host cells [START_REF] Shimoji | A 21-kDa surface protein of Mycobacterium leprae binds peripheral nerve laminin-2 and mediates Schwann cell invasion[END_REF][START_REF] Marques | Bacterial and host-derived cationic proteins bind alpha2-laminins and enhance Mycobacterium leprae attachment to human Schwann cells[END_REF][START_REF] Lima | Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae[END_REF][START_REF] Portugal | Characterization of two heparin sulphate-binding sites in the mycobacterial adhesin Hlp[END_REF]. Next, we then tested the capacity of recombinant M. leprae LBP/Hlp to bind collagen in a solid phase interaction assay. For this purpose, microplate wells were coated with M. leprae rLBP/Hlp and following a blocking step with BSA, increasing concentrations of biotinylated collagen I were added to the wells. The complexes were finally developed using a streptavidin-peroxidase conjugate. As shown in Fig. 5A, BSA displayed no significant collagen-binding activity for collagen I. In contrast, M. leprae rLBP/Hlp was demonstrated to bind collagen I in a dose-dependent and saturable manner.

Recombinant LBP/Hlp binds different collagen types via its C-terminal domain -The NaCl elution of M. bovis BCG LBP/Hlp from a chromatographic matrix bearing Fig. 3: effect of collagen pretreatment on the Mycobacterium bovis BCG cytoadherence. Exponentially-growing M. bovis BCG were pretreated or not with 50 µg/mL collagen I and then assayed for their interaction with A549 pneumocytes or U937 macrophages using a multiplicity of infection of 10. Colony forming units associated with target cells were counted by plating serial cell lysate dilutions on 7H11 agar. The data with standard deviations represent averages for quadruplicate experiments. covalently immobilized collagen I suggested that ionic interactions were more likely involved in the adhesin interaction with collagen. The C-terminal half of LBP/Hlp is highly positively charged and exhibits homology with the eukaryotic class H1 histones [START_REF] Lee | Upregulation of a histonelike protein in dormant Mycobacterium smegmatis[END_REF][START_REF] Prabhakar | Identification of an immunogenic histone-like protein (HLP-Mt) of Mycobacterium tuberculosis[END_REF]. In a recent investigation, we have used truncated recombinant M. leprae LBP/Hlp molecules corresponding to the N-terminal (residues 1-110, rLBP/Hlp-N) or the C-terminal (residues 111-200, rLBP/Hlp-C) domains and showed that the C-terminal half of the protein constitutes the major laminin and heparin-binding site of the adhesin [START_REF] Lima | Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae[END_REF][START_REF] Portugal | Characterization of two heparin sulphate-binding sites in the mycobacterial adhesin Hlp[END_REF]. We then tested if the C-terminal domain of LBP/Hlp was the collagen-interacting site of the protein. For this purpose, we assayed the collagen-binding activity of the M. leprae LBP/Hlp truncated forms which were previously shown to coat microplate wells with identical efficiencies [START_REF] Lima | Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae[END_REF]. Fig. 5A shows overlapping of the collagen Ibinding curves obtained with rLBP/Hlp and rLBP/Hlp-C, while no collagen I-binding activity was observed using rLBP/Hlp-N. This finding indicates that the interaction of LBP/Hlp with collagen I is mediated by the C-terminal half of the adhesin. In contrast, rLBP/Hlp-N displayed very limited binding capacity to collagen I. Finally, we next tested the capacity of full-length rLBP/Hlp and truncated forms to bind to other collagen types. As shown in Fig. 5B, the rLBP/Hlp and rLBP/Hlp-C were also able to significantly bind collagens III, IV and VI. These results suggest that, similarly to its interaction with heparin and laminin, LBP/Hlp binds collagen mainly through interactions involving the lysine-rich repeats present in the C-terminal domain of the adhesin.

DISCUSSION

Because specific interactions with host cells represent a crucial and early step in any infectious process, pathogenic microorganisms have evolved adhesins capable to bind membrane receptors or compounds present in the eukaryotic extracellular matrices [START_REF] Westerlund | Bacterial proteins binding to the mammalian extracellular matrix[END_REF]. Pathogenic mycobacteria synthesize a wide panel of adhesins involved in the bacterial interaction with epithelial cells and/or monocyte-derived professional phagocytes [START_REF] Hoppe | Identification of phosphatidylinositol mannoside as a mycobacterial adhesin mediating both direct and opsonic binding to nonphagocytic mammalian cells[END_REF][START_REF] Sidobre | Lipoglycans are putative ligands for the human pulmonary surfactant protein A attachment to mycobacteria. Critical role of the lipids for lectin-carbohydrate recognition[END_REF][START_REF] Diaz-Silvestre | The 19-kDa antigen of Mycobacterium tuberculosis is a major adhesin that binds the mannose receptor of THP-1 monocytic cells and promotes phagocytosis of mycobacteria[END_REF][START_REF] Kinhikar | Mycobacterium tuberculosis malate synthase is a laminin-binding adhesin[END_REF][START_REF] Ragas | The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A[END_REF][START_REF] Hickey | Mycobacterium tuberculosis employs Cpn60.2 as an adhesin that binds CD43 on the macrophage surface[END_REF]. Several proteinaceous mycobacterial adhesins have been demonstrated to recognize mammalian extracellular matrix components. Such an adhesive activity has been demonstrated for the proteins belonging to the antigen 85 complex and the fibronectin attachment protein, which both bind fibronectin [START_REF] Abou-Zeid | Genetic and immunological analysis of Mycobacterium tuberculosis fibronectin-binding proteins[END_REF][START_REF] Peake | Mechanism of interaction of the 85B secreted protein of Mycobacterium bovis with fibronectin[END_REF][START_REF] Zhao | Characterization of the fibronectin binding motif for a unique mycobacterial fibronectin attachment protein, FAP[END_REF], the HBHA which interacts with heparan sulphate-containing proteoglycans [START_REF] Delogu | Functional domains present in the mycobacterial hemagglutinin, HBHA[END_REF][START_REF] Pethe | Mycobacterial heparin-binding hemagglutinin and laminin-binding protein share antigenic methyllysines that confer resistance to proteolysis[END_REF] and the LBP/Hlp (also called MDP1) that binds laminin, heparan sulphate chains and HA [START_REF] Shimoji | A 21-kDa surface protein of Mycobacterium leprae binds peripheral nerve laminin-2 and mediates Schwann cell invasion[END_REF][START_REF] Aoki | Extracellular mycobacterial DNA-binding protein 1 participates in mycobacterium-lung epithelial cell interaction through hyaluronic acid[END_REF][START_REF] Lima | Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae[END_REF][START_REF] Lefrançois | Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/ histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease[END_REF].

Evidence strongly suggests that LBP/Hlp displays a bi-functional role in the biology of mycobacteria, acting both in nuclei acid metabolism [START_REF] Prabhakar | Identification of an immunogenic histone-like protein (HLP-Mt) of Mycobacterium tuberculosis[END_REF] and as an adhesin. In this regard, LBP/Hlp was initially characterized as a LBP, mediating the binding and invasion of SC by M. leprae [START_REF] Shimoji | A 21-kDa surface protein of Mycobacterium leprae binds peripheral nerve laminin-2 and mediates Schwann cell invasion[END_REF][START_REF] Marques | Bacterial and host-derived cationic proteins bind alpha2-laminins and enhance Mycobacterium leprae attachment to human Schwann cells[END_REF]. Recombinant LBP/Hlp was able to avidly bind laminin in solid phase assays and the exogenous addition of the protein significantly enhances bacterial binding to SC [START_REF] Marques | Bacterial and host-derived cationic proteins bind alpha2-laminins and enhance Mycobacterium leprae attachment to human Schwann cells[END_REF]. These data, together with the recent finding indicating that M. leprae actively expresses LBP/Hlp during the human infection [START_REF] Lima | Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae[END_REF], have reinforced the involvement of LBP/Hlp in the successful invasion and colonization of the peripheral nerve system by M. leprae. More recently, LBP/Hlp was also shown to play an important role in mycobacteria-lung epithelial cell interaction. Both M. tuberculosis and M. bovis BCG were shown to bind and invade A549 human lung epithelial cells through the interaction of LBP/Hlp with HA presence on the cell surface [START_REF] Aoki | Extracellular mycobacterial DNA-binding protein 1 participates in mycobacterium-lung epithelial cell interaction through hyaluronic acid[END_REF].

Collagenous proteins, which are predominant constituents of the mammalian extracellular matrix, can also function as receptors for the adhesion of microbial pathogens. The collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that are synthesized by both Gram-positive and Gram-negative bacteria constitute a large family of proteins involved in the bacterial attachment to host tissues [START_REF] Patti | MSCRAMM-mediated adherence of microorganisms to host tissues[END_REF]. Therefore, it is not surprising that collagen-binding MSCRAMMs may represent important virulence factors such as the CNA, YadA, Cpa and Dr adhesins, which are produced by Staphylococ-cus aureus, Yersinia enterocolitica, Streptococcus pyogenes and Escherichia coli, respectively and which play a role in the establishment and progression and/or persistence of the infection [START_REF] Gripenberg-Lerche | Role of YadA in arthritogenicity of Yersinia enterocolitica serotype O:8: experimental studies with rats[END_REF][START_REF] Selvarangan | Interaction of Dr adhesin with collagen type IV is a critical step in Escherichia coli renal persistence[END_REF][START_REF] Xu | Virulence potential of the staphylococcal adhesin CNA in experimental arthritis is determined by its affinity for collagen[END_REF][START_REF] Kreikemeyer | Streptococcus pyogenes collagen type Ibinding Cpa surface protein. Expression profile, binding characteristics, biological functions and potential clinical impact[END_REF]. Besides adhesins that bind collagen, more recently bacterial collagen-like surface proteins have been characterized and shown to play a role in microbial pathogenesis by mediating attachment to host cell collagen receptors [START_REF] Caswell | Identification of the first prokaryotic collagen sequence motif that mediates binding to human collagen receptors, integrins alpha2beta1 and alpha11beta1[END_REF]. So far, no studies have reported the interaction of mycobacteria with collagen. Here, we show for the first time that mycobacteria may interact directly with collagen proteins via the surface-exposed LBP/Hlp adhesin.

Evidence that mycobacteria can interact with collagen was initially obtained from the observation that collagen I was able to induce aggregation of bacterial cells. Images generated by SEM also showed collagen fibres interacting with the surface of bacterial cell. This interaction was confirmed by the observation that exogenous collagen was able to increase mycobacterial adherence to human type II A549 pneumocytes and U937 human macrophages in a saturable and dose-dependent manner. An affinity chromatography performed on collagen I-Sepharose allowed the purification of a collagen-binding component from BCG cell lysate that was identified by mass spectrometry as the LBP/Hlp protein. Furthermore, when produced in the recombinant form, BCG LBP/Hlp protein was able to bind collagen in microplate solid phase assays confirming that LBP/Hlp is also a collagen-binding protein. LBP/Hlp is a highly conserved protein detected on the surface of all mycobacterial species so far investigated [START_REF] Lefrançois | Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/ histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease[END_REF]. To show that LBP/Hlp is the major collagenbinding protein present on mycobacterial surface, we analyzed the interaction of collagen fibres with M. smegmatis Δhlp, the only hlp null mutant so far available in the context of mycobacterial species. This mutant has been characterized and used in several previous studies [START_REF] Lee | Upregulation of a histonelike protein in dormant Mycobacterium smegmatis[END_REF][START_REF] Marques | Bacterial and host-derived cationic proteins bind alpha2-laminins and enhance Mycobacterium leprae attachment to human Schwann cells[END_REF][START_REF] Biet | Mycobacterium smegmatis produces an HBHA homologue which is not involved in epithelial adherence[END_REF]. In contrast to the wt strain, M. smegmatis Δhlp was unable to interact and form large clumps when grown in the presence of collagen I, suggesting that LBP/Hlp represents a major component on bacterial surface mediating microbial interaction with collagen.

Mycobacterial LBP/Hlp is about twice the size of other bacterial Hlps. The C-terminal half of mycobacterial Hlp is absent from most other bacteria and has an unusual amino acid composition owing to a high alanine, lysine and proline content, which resembles the C-terminal region of eukaryotic class H1 histones [START_REF] Prabhakar | Identification of an immunogenic histone-like protein (HLP-Mt) of Mycobacterium tuberculosis[END_REF]. This highly positive domain was recently shown to mediate the interaction of LBP/Hlp with laminin and heparan sulphate and also with SC [START_REF] Lima | Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae[END_REF]. To map the region of LBP/Hlp responsible for the collagen binding activity, truncated versions of the adhesin corresponding to the N-terminal and C-terminal half of the protein were generated and tested in a solid phase collagen binding assay. These experiments clearly demonstrated that the lysine-rich C-terminal region holds most if not all of the collagen-binding properties of the protein.

Both full-length protein and the C-terminal domain were able to bind very similarly to collagen type I, while interaction with collagen III, IV and VI was partially mediated by the C-terminal domain. These data reinforce the idea that LBP/Hlp C-terminal domain is responsible for most if not all of the adhesive property of this protein.

The definition of the molecules used by M. tuberculosis and M. leprae to gain access and colonize different tissues of the human body may provide clues for the development of new therapeutic and/or prophylactic strategies to better control tuberculosis and leprosy. The capacity of LBP/Hlp to bind different collagen types here demonstrated reinforces its potential role as a virulence factor of pathogenic mycobacteria. The binding properties of LBP/ Hlp to multiple extracellular matrix components suggest that this protein may play an important role in distinct moments during the natural course of human infection. As previously suggested, early during infection, LBP/ Hlp might play, in conjunction with HBHA, a key role on bacterial attachment to respiratory epithelial cells [START_REF] Aoki | Extracellular mycobacterial DNA-binding protein 1 participates in mycobacterium-lung epithelial cell interaction through hyaluronic acid[END_REF]. We can also speculate on the involvement of LBP/Hlp in bacterial dissemination inside the human body that occurs both in leprosy and tuberculosis [START_REF] Pessolani | Systemic dissemination in tuberculosis and leprosy: do mycobacterial adhesins play a role?[END_REF]. During this process, the bacilli might need to interact with distinct basal membranes, which are essentially composed of laminin and collagen IV [START_REF] Kreis | Guidebook to the extracellular matrix and adhesion proteins[END_REF]. Finally, LBP/Hlp may also participate of the colonization of vulnerable tissues such as the peripheral nerve system by M. leprae through interaction mainly with laminin [START_REF] Shimoji | A 21-kDa surface protein of Mycobacterium leprae binds peripheral nerve laminin-2 and mediates Schwann cell invasion[END_REF][START_REF] Marques | Bacterial and host-derived cationic proteins bind alpha2-laminins and enhance Mycobacterium leprae attachment to human Schwann cells[END_REF] and the bones and joints by M. tuberculosis, which are rich in collagen I [START_REF] Kreis | Guidebook to the extracellular matrix and adhesion proteins[END_REF].

Fig. 1 :

 1 Fig. 1: Mycobacterium bovis BCG grown in the absence or the presence of collagen I. Bacilli grown in the absence (A) or the presence (B) of 25 µg/mL collagen I were fixed and observed by scanning electron microscopy. The arrow indicates collagen fibres that surround mycobacteria.

Fig. 2 :

 2 Fig.2: effect of exogenous collagen I on the Mycobacterium bovis BCG interaction with pneumocytes and macrophages. Exponentiallygrowing bacilli were incubated for 2 h with A549 type II human pneumocytes or with human U937 macrophages using a multiplicity of infection of 10 in the presence of increasing concentrations of collagen I. After washing to remove unbound mycobacteria, colony forming units associated with both cell lines were counted by plating serial cell lysate dilutions onto 7H11 agar. The data represent averages for quadruplicate experiments and standard deviation bars are shown.

Fig. 5 :

 5 Fig. 5: Mycobacterium leprae laminin-binding histone-like protein (LBP/Hlp) binds to different collagen types by its C-terminal domain. Microplate wells coated with 5 µg/mL of full-length M. leprae recombinant-LBP/Hlp, truncated N-terminal (rLBP/Hlp-N) or C-terminal (rLBP/Hlp-C) domains were incubated with increasing concentrations of biotinylated collagen I (A) or 40 µg/mL of collagens I, III, IV or VI (B). Binding is expressed in absorbency units at 490 nm. Data represent the mean +/-standard deviation of a typical experiment done in duplicate. Five experiments were performed with similar results. BSA: bovine serum albumin.

Fig

  Fig. 4A: sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting analyses of Mycobacterium bovis BCG collagenbinding protein. The Coomassie-blue staining of a polyacrylamide gel containing a clarified lysate of M. bovis BCG prior application onto a collagen-Sepharose matrix (Lane 1) and the salt-eluted material (Lane 2); B: recombinant M. bovis BCG laminin-binding histone-like protein (LBP/Hlp) binds collagen. Microplate wells coated with 1 µg/mL of collagen I were incubated with increasing concentrations of M. bovis BCG recombinant-LBP/Hlp. The wells were then incubated with the anti-LBP/Hlp monoclonal antibody 5G9 and, finally, with a rabbit anti-mouse IgG peroxidase conjugate; C: the Mycobacterium smegmatis wild-type (wt), but not the Δhlp strain, was able to form large clumps in the presence of collagen I. M. smegmatis wt and the mutant for the hlp gene (Δhlp) were grown in complete Middlebrook 7H9 broth supplemented or not with type 1 calf skin collagen at the concentration of 25 μg/mL. When cultures reached an OD 600nm of 1.8, mycobacteria were acid-fast stained by Kynion and visualized under light microscope with 400X magnification; BSA: bovine serum albumin.

ACKNOWLEDGEMENTS

To Dr John S Spencer, from the Colorado State University, for providing the 5G9 anti-rLBP/Hlp monoclonal antibody, to Elizabeth Pradel and Yves Dufrêne, for critical reading of the manuscript, to Jean-Pierre Tissier, for the technical assistance, and to the memory of Franco Dante Menozzi, who died in July 2005.

Scanning electron micrographs of Mycobacterium bovis BCG grown in the absence or the presence of collagen I. Mycobacteria grown in Sauton medium (Panel A) or in Sauton medium supplemented with 25 µg/mL collagen I (Panel B) were resuspended in phosphate buffered saline (PBS) and fixed for scanning electron microscopy (SEM) examination. A similar fixation step was applied to a collagen I solution prepared in PBS, followed by SEM observation (Panel C).