Roles for cell wall glycopeptidolipid in surface adherence and planktonic dispersal of Mycobacterium avium

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.

FIG. 1.

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

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.

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.

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.

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.