Actin Polymerization Drives Septation of Listeria monocytogenes namA Hydrolase Mutants, Demonstrating Host Correction of a Bacterial Defect

Abstract

The Gram-positive bacterial cell wall presents a structural barrier that requires modification for protein secretion and large-molecule transport as well as for bacterial growth and cell division. The Gram-positive bacterium Listeria monocytogenes adjusts cell wall architecture to promote its survival in diverse environments that include soil and the cytosol of mammalian cells. Here we provide evidence for the enzymatic flexibility of the murein hydrolase NamA and demonstrate that bacterial septation defects associated with a loss of NamA are functionally complemented by physical forces associated with actin polymerization within the host cell cytosol. L. monocytogenes ⌬namA mutants formed long bacterial chains during exponential growth in broth culture; however, normal septation could be restored if mutant cells were cocultured with wild-type L. monocytogenes bacteria or by the addition of exogenous NamA. Surprisingly, ⌬namA mutants were not significantly attenuated for virulence in mice despite the pronounced exponential growth septation defect. The physical force of L. monocytogenes-mediated actin polymerization within the cytosol was sufficient to sever ⌬namA mutant intracellular chains and thereby enable the process of bacterial cell-to-cell spread so critical for L. monocytogenes virulence. The inhibition of actin polymerization by cytochalasin D resulted in extended intracellular bacterial chains for which septation was restored following drug removal. Thus, despite the requirement for NamA for the normal septation of exponentially growing L. monocytogenes cells, the hydrolase is essentially dispensable once L. monocytogenes gains access to the host cell cytosol. This phenomenon represents a notable example of eukaryotic host cell complementation of a bacterial defect.

Host Actin Polymerization Tunes the Cell Division Cycle of an Intracellular Pathogen

“…Given that septation of a PG hydrolase mutant can be rescued by actin polymerization during infection (Alonzo et al, 2011) and that excess hydrolase can compensate for the loss of actin polymerization to shorten intracellular Lm division (Figures 3B and 3C), we speculate that Lm septation is directly or indirectly augmented by the mechanical stress associated with this process.…”

“…We focused on a potential role for ActA, a PrfA-controlled cell surface protein that is required for in vivo survival because it nucleates host actin to form tails that propel the bacterium from cell to cell (de las Heras et al, 2011; Kocks et al, 1992). Of note, host actin polymerization can correct intracellular cell division for a septation-deficient mutant (Alonzo et al, 2011). …”

“…An Lm mutant lacking the NamA (MurA) PG hydrolase forms chains and is defective for actin tail formation at early stages of cellular infection but replicates as single cells with actin tails at later time points (Alonzo et al, 2011). Cytochalasin D treatment abrogates this phenotype, suggesting that actin polymerization can rescue ΔnamA septation under certain circumstances.…”

“…The observation that ΔnamA chains are disrupted by actin polymerization (Alonzo et al, 2011) implied that the effects of actin polymerization and PG hydrolysis on cell division might be partially interchangeable. To test this idea, we first established a system for manipulating PG hydrolysis under broth and host cell conditions by lysozyme (Supplemental Experimental Procedures).…”

prfA -Like Transcription Factor Gene lmo0753 Contributes to l -Rhamnose Utilization in Listeria monocytogenes Strains Associated with Human Food-Borne Infections

“…Construction of lmo0753 deletion mutants and complements. L. monocytogenes gene deletions in 10403S and EGDe were constructed using methods described by Alonzo et al (15). All primers used are listed in Table 2.…”