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Applied and Environmental Microbiology, December 2006, p. 7554-7558, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01633-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Roles for Cell Wall Glycopeptidolipid in Surface Adherence and Planktonic Dispersal of Mycobacterium avium{triangledown}

Robert Freeman,1 Henriette Geier,2 Kris M. Weigel,1 Julie Do,1 Timothy E. Ford,2 and Gerard A. Cangelosi1*

Seattle Biomedical Research Institute, Seattle, Washington,1 Department of Microbiology, Montana State University, Bozeman, Montana2

Received 13 July 2006/ Accepted 25 September 2006


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ABSTRACT
 
The opportunistic pathogen Mycobacterium avium is a significant inhabitant of biofilms in drinking water distribution systems. M. avium expresses on its cell surface serovar-specific glycopeptidolipids (ssGPLs). Studies have implicated the core GPL in biofilm formation by M. avium and by other Mycobacterium species. In order to test this hypothesis in a directed fashion, three model systems were used to examine biofilm formation by mutants of M. avium with transposon insertions into pstAB (also known as nrp and mps). pstAB encodes the nonribosomal peptide synthetase that catalyzes the synthesis of the core GPL. The mutants did not adhere to polyvinyl chloride plates; however, they adhered well to plastic and glass chamber slide surfaces, albeit with different morphologies from the parent strain. In a model that quantified surface adherence under recirculating water, wild-type and pstAB mutant cells accumulated on stainless steel surfaces in equal numbers. Unexpectedly, pstAB mutant cells were >10-fold less abundant in the recirculating-water phase than parent strain cells. These observations show that GPLs are directly or indirectly required for colonization of some, but by no means all, surfaces. Under some conditions, GPLs may play an entirely different role by facilitating the survival or dispersal of nonadherent M. avium cells in circulating water. Such a function could contribute to waterborne M. avium infection.


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INTRODUCTION
 
Members of the Mycobacterium avium complex (MAC), a group of closely related species and subspecies, are commonly isolated from water, food, soil, plants, and other samples. MAC species are associated with disease in birds and mammals, and some cause disease in susceptible humans. Sources of exposure include drinking water, spas, and soil. Mycobacteria are significant inhabitants of biofilms in these environments. They have been found in biofilm samples taken from water distribution systems, dental units, and medical devices at frequencies ranging from 69% to 95% of samples tested (1, 11, 12, 25). Among the largest studies of mycobacteria in biofilms was that of Falkinham et al. (11). They sampled biofilms from posttreatment water pipes and customer water meters in eight U.S. cities. Mycobacteria were recovered from 69% of the samples and 100% of the sites. Slow-growing mycobacteria accounted for >90% of biofilm isolates. MAC species were the most common group recovered, accounting for 135 of the 267 (51%) individual Mycobacterium isolates from biofilm samples. In laboratory experiments, biofilm formation by MAC species has been correlated with chlorine resistance (24) and enhanced bronchial epithelial cell invasion (26). Despite its potential significance for human health, little is known about biofilm formation by the MAC.

The cell walls of some Mycobacterium species have glycopeptidolipids (GPLs) that share a lipotetrapeptide core consisting of fatty acyl-NH-D-phenylalanine-D-allothreonine-D-alanine-L-alaninol (7). The alaninyl C terminus is rhamnosylated, and the allothreonine residue is linked to a 6-deoxy-L-talose. In the MAC, the latter is further modified with variable, highly antigenic oligosaccharide structures that form serovar-specific GPLs [ssGPLs]. Situated on the outer cell wall surface, ssGPLs account in part for the characteristic smooth, wet appearance of MAC colonies. Over the course of subculture, spontaneous "rough"-colony-type variants that have missing or altered ssGPLs frequently arise. At least some rough-colony-type variants result from spontaneous mutations within a cluster of ssGPL biosynthetic and translocation genes (2, 10).

Little is known about the function of GPLs in infection by or free-living growth of mycobacteria. They have been implicated in virulence, possibly through inhibition of macrophage activation (14, 23). They have also been correlated with mycobacterial biofilm formation. The core GPL was shown to be required for biofilm formation by the fast-growing model organism M. smegmatis (22). In M. avium subspecies hominissuis, a MAC subspecies that is opportunistically pathogenic to humans (18), a single biofilm-defective mutant was found to have a transposon insertion into pstAB (also known as nrp and mps), which encodes the nonribosomal peptide synthetase that generates the lipotetrapeptide core (27). The GPL content was also correlated with strain-to-strain variation in biofilm formation by M. avium subspecies hominissuis (6). These studies used the high-throughput 96-well model for quantifying adherence to polyvinyl chloride (PVC) surfaces. More recently, GPLs were correlated with the ability of a colony-type variant of M. abscessus to form biofilms on the polystyrene pegs of the Calgary Biofilm Device (13).

Although other cell wall lipids have also been implicated in mycobacterial biofilms (8, 20), a consensus is emerging that GPLs play a role. In order to test this consensus in greater depth, we examined the abilities of replicate, independently isolated pstAB mutants of M. avium subspecies hominissuis to form biofilms on diverse surfaces in three model systems. These models demonstrated that the core GPL is required for adherent accumulation of M. avium subspecies hominissuis on some, but by no means all, surfaces. Under some conditions, it may play an entirely different role by facilitating the survival or dispersal of nonadherent M. avium subspecies hominissuis cells in circulating water.


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MATERIALS AND METHODS
 
Bacterial strains and culture conditions.
M. avium subspecies hominissuis strain HMC02 is a clinical isolate from an anonymous patient in Seattle, Washington. The organism was grown on Middlebrook 7H10 agar plates (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase enrichment (BBL), 0.5% glycerol, and 100 µg/ml Congo red or on Middlebrook 7H9 broth medium supplemented with 10% albumin-dextrose-catalase enrichment and 0.2% glycerol. Mutants with transposon insertions into pstAB were isolated from an EZ-TN (KAN-2) transposon insertion library described previously (5, 21) by screening for colony morphotype, as described in Results.

Biofilm formation in 96-well PVC plates.
Bacterial cultures were grown in biofilm medium (22) consisting of M63 salts minimal medium (Amresco, Inc., Solon, OH) supplemented with 2% glucose, 0.5% Casamino Acids, 1 mM MgSO4, and 0.7 mM CaCl2. Upon reaching an optical density at 600 nm (OD600) of approximately 0.4, they were centrifuged and resuspended to an OD600 of 0.2 in biofilm medium. Cell suspensions (200 µl per well) were added to the wells of 96-well, U-bottom PVC microtiter plates (Falcon no. 353911). The plates were sealed with Breathe-Easy Sealing Membranes (Sigma no. Z380059) and incubated without agitation at 37°C for 4 weeks. The wells were vigorously washed with flowing tap water and then stained with 1% crystal violet solution (50 µl per well) for 20 min. The wells were again rinsed and allowed to dry, and the dye was solubilized with 95% ethanol (100 µl per well). The ethanol extracts were transferred to UV flat-bottom plates for reading the absorbance at 570 nm. At least four wells with biofilm medium but no bacteria were used as negative controls.

Biofilm formation in chamber slides.
For visual observation of surface attachment, sterile eight-well-per-chamber plastic (Permanox) and glass chamber slides were obtained from Fisher Scientific (catalog numbers 12-565-18 and 12-565-22). Bacterial inocula were grown in 7H9 broth to an OD600 of 0.5 to 1.0, centrifuged, and resuspended in biofilm medium to an OD600 of 0.2 as described above for the 96-well model. Suspensions (200 µl) were added in duplicate to slide chambers and covered. The slides were incubated at 37°C in a humidified incubator for up to 4 weeks. In order to replace water lost to evaporation, autoclaved water was added to the chambers every 7 to 10 days to restore the original volume. At 3-day intervals throughout the 4-week incubation period, slides (one per time point) were analyzed by microscopy as follows. A pipette was used to remove medium with unattached cells and to vigorously wash the slide surfaces with autoclaved deionized water. Adherent cells were stained for 30 min with 10x SYBR green in Tris-EDTA buffer. SYBR green is a cell-permeant fluorescent DNA stain (Invitrogen no. S-7563). After additional washes to remove unincorporated stain, bacterial adherence to the slide surfaces was visualized by fluorescence microscopy with a fluorescein isothiocyanate filter set. Six representative fields were photographed per chamber.

Accumulation of viable M. avium subspecies hominissuis on stainless steel surfaces and in recirculating water.
The recirculating system is shown in Fig. 1. Water circulation was powered by a Masterflex pump head (Cole-Parmer model 7518-00). The tubing used was Masterflex Norprene (06402-25), with an inner diameter of 0.5 mm. The reservoir vessel was filled with 500 ml of sterile Milli-Q water and connected to both sides of a 23-in. glass tube with an inner diameter of 1.5 cm. All connections were made with silicon stoppers. Contained within each tube were four stainless steel coupons with dimensions of 1.3 by 8.0 cm, held together by stainless steel wire. After assembly, the entire system was autoclaved.


Figure 1
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  		FIG. 1. Recirculating model system. A bank of three replicate systems is shown, each with four coupons housed in a glass tube.

Cells taken from a 7H10 plate were suspended in 1 ml sterile Milli-Q purified water. These suspensions were added to 2-ml screw-cap tubes containing 600 to 800 µl glass beads (2-mm diameter). To ensure uniform dispersal of variably flocculent M. avium subspecies hominissuis cell suspensions, the cells were mechanically dispersed by agitation with a Biospec Products Mini-Beadbeater. Bead beating was performed at the lowest power in two 15-s bursts. The density of each culture was read by using a Klett Photoelectric Colorimeter Model 800-3 and normalized to 75 Klett units (equivalent to an OD660 of approximately 0.15) prior to inoculation. Eight milliliters of each adjusted cell suspension was then injected into a recirculation system. The pump was adjusted to a flow rate of 38 ml/min, and the system was allowed to run at ambient temperature for 2 weeks.

At the end of the incubation period, four coupons and two water samples were taken for analysis as follows. Glass tubes were aseptically removed from the system and transferred to a hood. The coupons were pulled out of the cartridge and extensively rinsed to remove nonadherent cells. Adherent cells were scraped off of the coupons with a rubber policeman and suspended in 10 ml of sterile water. After bead beating to disperse clumps as described above, replicate samples were serially diluted onto 7H10 agar plates. Ten replicates were plated per dilution. Bacteria in the water (planktonic) phase of the samples in glass tubes were also analyzed. Two 1-ml samples per tube were serially diluted onto 7H10 agar in replicates of 10, as described above.


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RESULTS
 
Isolation of pstAB mutants of M. avium subspecies hominissuis.
As part of an investigation aimed at understanding the reversible morphotypic switches of M. avium subspecies hominissuis, an EZ-TN transposon library was generated in a smooth, white opaque (WO) colony-type variant of M. avium subspecies hominissuis strain HMC02 (HMC02-WO). The WO colony type is associated with increased virulence and multidrug resistance relative to opaque colonies that stain red (RO) on Congo red agar (4, 19). Mutants with altered colony morphology were isolated and characterized (5, 15, 21).

Twelve mutants that formed red, rough colonies on Congo red agar were isolated. Transposon insertions were mapped by randomly primed PCR (21). All 12 mutants had insertions into contiguous genes originally described by Eckstein et al. (9) as pstA and pstB (NCBI loci AAD44233 and AAD44234). They are orthologous (Expect value, 0.0) to the 5' and 3' halves of mps (AAT01806), a single large gene in M. smegmatis. In the finished genome sequence of M. avium subspecies paratuberculosis strain K10, their ortholog is also a single large locus, MAP1242 (AAS03558.1). Mps is a peptide synthetase that was shown to catalyze the synthesis of the four-residue peptide of the core GPL in M. smegmatis (3). It has 5,990 amino acids, about equal in size to the combined PstA (2,552 amino acids) and PstB (3,445 amino acids) proteins of M. avium subspecies hominissuis. In silico analysis of functional modules suggested that PstA plus PstB of M. avium subspecies hominissuis and Mps of M. smegmatis perform similar functions and differ from each other mainly by virtue of their separation into two adjacent genes in some MAC strains (15). The pstAB locus mutagenized in this study is the same one that was mutationally correlated with biofilm formation by M. avium subspecies hominissuis strain A5 (27).

The mapped positions of transposon insertions in pstA and pstB are shown in Fig. 2. Through multiple passages on agar media, no smooth-colony revertants were observed, consistent with a stable GPL-negative phenotype. Three mutants with insertions into pstA, designated 13.1641, 15.1899, and 20.2439, were chosen for further analysis. Each was generated in a separate mutagenesis procedure.


Figure 2
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  		FIG. 2. Positions of EZ-TN transposon insertions in pstA and pstB. The positions of genes and insertion elements (gray triangles) are based on the sequence of the region in M. avium subspecies hominissuis strain 2151 (NCBI accession no. AF143772). The positions of EZ-TN transposon insertions (black circles) in pstAB are shown. Insertions discussed in the text are labeled by mutant strain designations. Each labeled insertion was generated in a separate mutagenesis procedure.

Biofilm formation in a static model with a PVC surface.
Two pstA mutants, 13.1641 and 15.1899, were assessed for biofilm formation in the static 96-well PVC model. After 4 weeks of incubation at 37°C, followed by vigorous washing to remove nonadherent cells, the mutants showed markedly reduced surface adherence relative to the WO parent clone (Fig. 3). Previous mutational studies had associated GPLs with biofilm formation under these conditions (22, 27). This finding confirms those reports and extends them by examining independently isolated mutants of M. avium subspecies hominissuis.


Figure 3
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  		FIG. 3. Adherence of M. avium subspecies hominissuis morphotypic variants and mutants to PVC microwells. Adherence was measured as retention of crystal violet-stained material on well surfaces after vigorous washing. Crystal violet was extracted into ethanol and quantified by absorbance at 570 nm. All clones examined were natural morphotypic variants (WO, RO, WT, and RT) of strain HMC02 or EZ-TN transposon mutants (13.1641 and 15.1899) of the WO variant of strain HMC02. The data are means and standard deviations of eight wells per strain. Replicate experiments yielded identical results.

Naturally occurring smooth RO, WO, red transparent (RT), and white transparent (WT) morphotypic variants of M. avium subspecies hominissuis strain HMC02 were also tested for their abilities to form biofilms under these conditions. The strongest binding was exhibited by the WT variant, which is also the most virulent form of this strain (4, 19). One morphotype, RO, exhibited markedly lower binding than the rest (Fig. 3).

Biofilm formation in a static model with Permanox and silanized glass surfaces.
In order to visualize adherence microscopically, wild-type (HMC02-WO) and pstA mutant (13.1641 and 15.1899) cells were inoculated into chamber slides in biofilm medium and incubated without agitation for 4 weeks, as in the 96-well assay. The chamber slides had wettable Permanox or silanized glass surfaces. Microscopic analysis at ~3-day intervals revealed consistently greater binding to Permanox slides. Maximum binding was seen 14 to 23 days postinoculation.

After vigorous rinsing, the pstA mutants 13.1641 and 15.1899 remained bound to both surfaces in numbers that appeared to be at least as great as those of the parent strain (Fig. 4). However, mutant and wild-type cells exhibited different binding morphologies. While the parent strain formed uniform monolayers of single cells and small clusters, the mutants exhibited much more flocculent patterns of binding (Fig. 4).


Figure 4
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  		FIG. 4. Biofilm formation on Permanox and silanized glass chamber slides. The attachment of the parent strain, HMC02-WO, and mutants 13.1641 and 15.1899 to slide surfaces was examined microscopically as described in the text. Replicate experiments yielded identical results.

Biofilm formation on a stainless steel surface in a recirculating-water distribution model.
In order to more faithfully model the conditions of a drinking water distribution system, a laboratory-based system was used to quantify adherence to stainless steel surfaces under recirculating water (Fig. 1). Replicate systems were inoculated with the parent HMC02-WO clone and three pstA mutants, 13.1641, 15.1899, and 20.2439. At the conclusion of the 14-day incubation period, recirculating, as well as adherent, bacteria were quantified by plating and colony counting. The parent and mutant strains did not differ quantitatively in their adherence to stainless steel coupons under these conditions. However, a consistent quantitative difference was seen in the recirculating phase, where the parent strain was found in ≥10-fold greater numbers than the mutants after 14 days (Fig. 5). Similar results (not shown) were obtained in a replicate experiment conducted on the same strains and in a third experiment conducted on the parent strain, HMC02-WO, and two pstB mutants, 6.418 and 24.3411 (map positions are shown in Fig. 2). In all cases, mutant and wild-type adherences to coupons were indistinguishable (within 2 standard deviations), while wild-type colony counts in the recirculating-water phase were ≥10-fold greater than those of the mutants (P < 0.001 by Student's t test).


Figure 5
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  		FIG. 5. Accumulation on stainless steel coupons and in recirculating water. Viable bacteria adhering to surfaces and recirculating in water were quantified as described in the text. The data are means and standard deviations of four coupons or two water samples per clone, as described in Materials and Methods.


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DISCUSSION
 
MAC species are significant opportunistic pathogens of humans and animals and among the most common Mycobacterium species isolated from biofilms in nature (11). Previous studies have implicated GPLs in biofilm formation by a MAC species, M. avium subspecies hominissuis, and by other mycobacteria (6, 13, 22, 27). In order to confirm and extend this correlation, we analyzed multiple, independently isolated pstAB mutants of M. avium subspecies hominissuis in three different model systems. The use of diverse models revealed a more complex picture than that which has arisen from studies based on single model systems.

The requirement for pstAB in binding of M. avium subspecies hominissuis to PVC microwells was confirmed. The results also indicated that the colony type impacts biofilm formation, with white and transparent variants adhering to PVC wells in greater amounts than their red and opaque counterparts. Interestingly, white and transparent variants are isolated more frequently from patients, and are more virulent in animal models, than red and opaque variants (4, 19). The ability of M. avium subspecies hominissuis cells to form biofilms on PVC plates has been correlated with their ability to invade bronchial epithelial cells (26). It is conceivable that the morphotype plays a role in this correlation.

Past studies conducted on M. smegmatis (16, 17, 22) and M. abscessus (13) correlated the ability to form biofilms on PVC and polystyrene with the capacity to spread across soft agar surfaces (sliding motility). Our results did not confirm this correlation in M. avium subspecies hominissuis. The RO variant of strain HMC02, which adhered very poorly to PVC microwells (Fig. 2), was previously shown to exhibit much greater sliding motility than its WO, RT, and WT counterparts (4).

The alternative models used in this study yielded results that differed from those seen in PVC microwells. The pstAB mutants adhered at least as well as the parent strain to Permanox and silanized glass chamber slides, indicating that the core GPL is not required for adhesion to these surfaces. However, a clear morphological phenotype was observed, with the mutants binding in large clumps rather than as isolated cells in a monolayer. The reasons for this are not known. The waxier surfaces of rough cells may not interact well with the wettable Permanox and silanized glass surfaces, resulting in a preferential interaction of cells with each other. Despite this, clumps of mutant bacteria adhered strongly to the slide surfaces and resisted removal under vigorous wash conditions. Due to high-frequency morphotypic switching, M. avium subspecies hominissuis cell populations tend to be heterogeneous. It is possible that subpopulations of mutant cells were able to adhere to the surfaces and were then bound by other cell types whose surface properties caused them to adhere preferentially to other M. avium subspecies hominissuis cells.

The recirculating system was designed to model drinking water distribution conditions. This model had limitations, including the use of a single-species inoculum and purified water in the recirculating phase (used to maximize reproducibility). Stainless steel surfaces are found in point-of-use fixtures but are not the major habitat in water distribution pipes, which typically have PVC surfaces overlaid with multispecies bacterial biofilms. Nonetheless, the recirculating model is in many ways more authentic than static models used in previous analyses, and it yielded unexpected results. Mutant and wild-type cells bound equally well to the stainless steel coupons, indicating that the core GPL is dispensable in the primary stages of biofilm formation under these conditions. However, the mutants were present in relatively small numbers in the recirculating-water (planktonic) phase. The basis for this observation is not known. Explanations could include (i) inefficient detachment of mutant cells from biofilms due to enhanced cell-to-cell interactions; (ii) poor survival in purified water for nutritional, structural, or other reasons; and (iii) inefficient suspension in water due to hydrophobic surface properties.

Biofilm formation is important to a pathogen's ability to colonize environmental reservoirs. However, ultimately it is detached cells that are ingested or inhaled by human and animal hosts. A requirement for GPLs in planktonic dispersal, if confirmed in additional models, would constitute an important role for these cell wall components in colonization of new environments and also in ingestion of MAC cells by humans.


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ACKNOWLEDGMENTS
 
We are grateful to Luiz Bermudez for helpful discussions.

This work was supported by grant R21-AI061006 from The National Institutes of Health and grant DAAD 19-03-1-0198 from the Army Research Office, overseen by Sherry Tove, Chief, Microbiology and Biodegradation, Life Sciences Division. Grant 833030010 from the U.S. Environmental Protection Agency supported the writing and submission of the manuscript.

Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the Army Research Office.


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FOOTNOTES
 
* Corresponding author. Mailing address: Seattle Biomedical Research Institute, 307 Westlake Avenue N, Suite 500, Seattle, WA 98109. Phone: (206) 256-7200. Fax: (206) 256-7229. E-mail: Jerry.Cangelosi{at}sbri.org. Back

{triangledown} Published ahead of print on 29 September 2006. Back


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Applied and Environmental Microbiology, December 2006, p. 7554-7558, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01633-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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