Conflicting roles for a cell surface modification in Salmonella

Abstract

Chemical modifications of components of the bacterial cell envelope can enhance resistance to antimicrobial agents. Why then are such modifications produced only under specific conditions? Here, we address this question by examining the role of regulated variations in O-antigen length in the lipopolysaccharide (LPS), a glycolipid that forms most of the outer leaflet of the outer membrane in Gram-negative bacteria. We determined that activation of the PmrA/PmrB two-component system, which is the major regulator of LPS alterations in Salmonella enterica serovar Typhimurium, impaired growth of Salmonella in bile. This growth defect required the PmrA-activated gene wzz(st), which encodes the protein that determines long O-antigen chain length and confers resistance to complement-mediated killing. By contrast, this growth defect did not require the wzz(fepE) gene, which controls production of very long O-antigen, or other PmrA-activated genes that mediate modifications of lipid A or core regions of the LPS. Additionally, we establish that long O-antigen inhibits growth in bile only in the presence of enterobacterial common antigen, an outer-membrane glycolipid that contributes to bile resistance. Our results suggest that Salmonella regulates the proportion of long O-antigen in its LPS to respond to the different conditions it faces during infection.

Fig. 1

Multiple environmental signals promote LPS modifications through PmrA-activated gene products. The phosphorylated form of PmrA activates expression of genes encoding proteins that mediate covalent modifications of the LPS. Fe³⁺ and acidic pH each can activate the sensor PmrB, which directly phosphorylates PmrA. Low Mg²⁺, cationic antimicrobial peptides, and acidic pH each can activate the PhoQ/PhoP two-component system, which promotes the phosphorylated form of the PmrA protein through the connector protein PmrD.

Fig. 2

Long O-antigen polysaccharide hinders growth in bile. A. Inactivation of wzz_st restores growth to the pmrA505 strain in bile. Wild-type [EG9493], pmrA505 [EG9492], and pmrA505 wzz_st [JM55, JM56] isogenic Salmonella strains were streaked onto plates containing N-minimal medium supplemented with 60 µM MgCl₂ and the indicated concentration of ox bile. B. Complementation of wzz_st mutant. Bacterial strains (pmrA505 + vector [JM61], pmrA505 wzz_st + vector [JM59], and pmrA505 wzz_st + pwzz_st [JM60]) were streaked onto plates containing N-minimal medium supplemented with ampicillin, 60 µM MgCl₂, the indicated concentration of ox bile, and isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce expression of wzz_st. C. wzz_fepE is not responsible for hindered growth of the pmrA505 strain in bile. Wildtype [EG9493], pmrA505 [EG9492], pmrA505 wzz_st [JM55], pmrA505 wzz_fepE [JM73, JM80], and pmrA505 wzz_fepE wzz_st [JM81, JM82] isogenic Salmonella strains were streaked onto plates containing N-minimal medium supplemented with 60 µM MgCl₂ and the indicated concentration of ox bile. Plates were incubated at 37 °C for 48 h before photography.

Fig. 3

PmrA-inducing conditions diminish growth of wild-type Salmonella in bile. Wild-type [14028s], wzz_st [EG14928], pmrA [EG13307], pmrA rcsB [EG13310], and pmrA rcsB wzz_st [JM160] isogenic Salmonella strains were streaked onto plates containing N-minimal medium supplemented with 60 µM MgCl₂, 100 µM FeSO₄, and the indicated concentration of ox bile.

Fig. 4

The ability of long O-antigen to impair growth in bile requires ECA. Wild-type [14028s], wzz_st [EG14928, EG14930], wecB [MP118], wecB wzz_st [JM146, JM147], ompC [EG16478], and ompC wzz_st [JM132] isogenic Salmonella strains were streaked onto plates containing N-minimal medium supplemented with 60 µM MgCl₂, 100 µM FeSO₄, and the indicated concentration of ox bile.

Fig. 5

wzz_st confers serum resistance but is detrimental for growth in bile. Shown are OD₆₀₀ values from wild-type (14028s) or wzz_st (EG14930) isogenic Salmonella strains that were treated with PBS, PBS supplemented with 10% human serum, or PBS supplemented with 10% ox bile. Data indicate the mean, and error bars show the standard deviation from three independent experiments.