Critical role of dispensable genes in Mycoplasma agalactiae interaction with mammalian cells

Abstract

Mycoplasmas are minimal bacteria whose genomes barely exceed the smallest amount of information required to sustain autonomous life. Despite this apparent simplicity, several mycoplasmas are successful pathogens of humans and animals, in which they establish intimate interactions with epithelial cells at mucosal surfaces. To identify biological functions mediating mycoplasma interactions with mammalian cells, we produced a library of transposon knockout mutants in the ruminant pathogen Mycoplasma agalactiae and used this library to identify mutants displaying a growth-deficient pheonotype in cell culture. M. agalactiae mutants displaying a 3-fold reduction in CFU titers to nearly complete extinction in coculture with HeLa cells were identified. Mapping of transposon insertion sites revealed 18 genomic regions putatively involved in the interaction of M. agalactiae with HeLa cells. Several of these regions encode proteins with features of membrane lipoproteins and/or were involved in horizontal gene transfer with phylogenetically distant pathogenic mycoplasmas of ruminants. Two mutants with the most extreme phenotype carry a transposon in a genomic region designated the NIF locus which encodes homologues of SufS and SufU, two proteins presumably involved in [Fe-S] cluster biosynthesis in Gram-positive bacteria. Complementation studies confirmed the conditional essentiality of the NIF locus, which was found to be critical for proliferation in the presence of HeLa cells and several other mammalian cell lines but dispensable for axenic growth. While our results raised questions regarding essential functions in mycoplasmas, they also provide a means for studying the role of mycoplasmas as minimal pathogens.

FIG. 1.

Growth and survival of M. agalactiae under cell culture conditions. Serial dilutions from mycoplasma stocks (A) or defined dilutions (B) were inoculated to HeLa cells seeded at a density of 2 × 10⁴ cells per cm² in DMEM-based medium (DMEM HeLa), DMEM-based medium alone (DMEM), DMEM-based medium preincubated with HeLa cells (DMEM HeLa SN), or DMEM-based medium supplemented with 10% Aluotto broth (DMEM Aluotto). Cultures were incubated at 37°C under 5% CO₂, and mycoplasma titers (log CFU/ml) were determined by CFU titrations following one freeze-thaw cycle of to disrupt HeLa cells. The data are the means of at least three independent assays. Standard deviations are indicated by error bars.

FIG. 2.

Characterization of M. agalactiae mutants displaying altered growth in cell culture. Mutants were designated according to transformation and clone numbers (e.g., T05.081 designates clone 81 isolated from transformation T05). PG2 and T08.101 refer to the parental strain and the control mutant, respectively. Group A mutants were selected from the mutant library by high-throughput screening on HeLa cells, as described in the Results section. Mycoplasma titers (log CFU/ml) at 48 h in Aluotto broth (open bars) and at 72 h in cell culture (solid bars) are compared. The data are the means of at least three independent assays. Standard deviations are indicated by error bars. Transposon insertion sites were determined by direct sequencing of genomic DNA, and their positions were defined based on the published sequence (NC_009497). The orientation of the inserted sequences is indicated in parenthesis. Mutants sharing an identical insertion are indicated. CDSs found disrupted in M. agalactiae mutants are indicated by the mnemonic codification (45). Noncoding regions are indicated. CDSs involved in horizontal gene transfer between M. agalactiae and mycoplasmas of the mycoides cluster (45) are designated by asterisks (*). For each CDS, the relative position and orientation of the inserted transposon are indicated (CDS position). Hypothetical proteins (HP) have no homologs outside the M. agalactiae species. Conserved hypothetical proteins (CHP) share sequence similarity with proteins of unknown function identified in mollicutes or other bacteria. Several insertion sites mapped within a 20-kb locus that contains a vestige of an integrative conjugative element (ICEA).

FIG. 3.

Clonality and stability of M. agalactiae NIF mutant populations. (A) The genomic regions surrounding the transposon insertion site in mutants T07.82 and T07.134 were analyzed by PCR amplification to detect the presence of contaminating transposon-free sequences. PCR1 and PCR2 were performed using the primer pair 86768F (5′-TCAGCCGACATTATTCATGG-3′) and 87272R (5′-CACCGGCTTTTAATTTTTGC-3′) and the pair 88037F (5′-AGGGTTTCGCTAGGGGTTTA-3′) and 88595R (5′-CTGTGCGCGCTTACAAAGTA-3′), respectively. (B) The PCR1 product (505 bp) amplified from mutant T07.134 populations at passages 2 (7.134p2) and 3 (7.134p3) was not detected upon amplification of the corresponding populations of mutant T07.82 (7.82p2 and 7.82p3). The opposite result was observed for the PCR2 product (558 bp). These negative results suggest the absence of detectable contaminating sequences in all the populations tested. M1, molecular weight markers (SmartLadder; Eurogentec). (C) Southern blot analysis of genomic DNA derived from NIF mutant T07.82 (7.82) at passages 1, 10, and 20 in selective (Gm) or nonselective medium. Genomic DNA was digested by HindIII and Southern blot hybridized with DIG-labeled amplicons derived from plasmid pMT85. M2, molecular weight markers (1 Kb DNA ladder; Promega).

FIG. 4.

Comparative growth of M. agalactiae NIF mutants under cell culture conditions. M. agalactiae parental strain PG2, the control mutant T08.101 (8.101), and NIF mutants T07.082 (7.82) and T07.134 (7.134) were assessed for survival over a 72-h incubation in DMEM-based medium (DMEM) or in coculture with a number of mammalian cell lines including HeLa, goat fibroblast (TIGEF), goat epithelial (TIGMEC), bovine turbinate (TB), and bovine esophageal (KOP) cells. The data are the means of two or three independent assays. Standard deviations are indicated by error bars.

FIG. 5.

Complementation of M. agalactiae NIF mutants. Schematic representation of the plasmid pO/T-NIF used for complementation studies (A). The NIF locus was cloned under the control of the P40 protein promoter region. M. agalactiae parental strain PG2, mutants T07.082 (7.82) and T07.134 (7.134), and mutants transformed with plasmid pO/T-NIF (7.82 NIF and 7.134 NIF) or the control plasmid p21-1miniO/T (7.82 OT and 7.134 OT) were assessed for colony development on Aluotto, 10% horse red blood cell (RBC) lysates, or 5% sheep blood agar plates (B) and for survival over a 72-h incubation in DMEM-based medium (DMEM) or in coculture with HeLa, goat fibroblast (TIGEF), or goat epithelial (TIGMEC) cells (C). The data are the means of at least three independent assays. Standard deviations are indicated by error bars. Serial dilutions of mycoplasma stocks were spotted on blood agar plates, and colony development was observed after 4 to 6 days of incubation at 37°C.