OmpR and Prc contribute to switch the Salmonella morphogenetic program in response to phagosome cues.

Salmonella enterica serovar Typhimurium infects eukaryotic cells residing within membrane-bound phagosomes. In this compartment, the pathogen replaces the morphogenetic penicillin-binding proteins 2 and 3 (PBP2/PBP3) with PBP2SAL /PBP3SAL , two proteins absent in Escherichia coli. The basis for this switch is unknown. Here, we show that PBP3 protein levels drop drastically when S. Typhimurium senses acidity, high osmolarity and nutrient scarcity, cues that activate virulence functions required for intra-phagosomal survival and proliferation. The protease Prc and the transcriptional regulator OmpR contribute to lower PBP3 levels whereas OmpR stimulates PBP2SAL /PBP3SAL production. Surprisingly, despite being essential for division in E. coli, PBP3 levels also drop in non-pathogenic and pathogenic E. coli exposed to phagosome cues. Such exposure alters E. coli morphology resulting in very long bent and twisted filaments indicative of failure in the cell division and elongation machineries. None of these aberrant shapes are detected in S. Typhimurium. Expression of PBP3SAL restores cell division in E. coli exposed to phagosome cues although the cells retain elongation defects in the longitudinal axis. By switching the morphogenetic program, OmpR and Prc allow S. Typhimurium to properly divide and elongate inside acidic phagosomes maintaining its cellular dimensions and the rod shape.

An alternative penicillin-binding protein involved in Salmonella relapses following ceftriaxone therapy.

BACKGROUND: Salmonella causes intracellular infections in humans. Besides quinolones, third generation cephalosporins are first line drugs used for salmonellosis therapy. An unresolved anomaly of this practice involves high relapse rates associated to quinolone- or cephalosporin-susceptible Salmonella isolates in patients that are discharged clinically following initial recovery. Reduced drug accessibility to intracellular locations has been hypothesized to impair pathogen eradication although supporting evidence is lacking in vivo. Here, we uncover a novel penicillin-binding protein as the first Salmonella factor likely contributing to relapse following beta-lactam, mainly ceftriaxone, therapy. METHODS: We used Salmonella enterica serovar Typhimurium mutants lacking the alternative penicillin-binding proteins PBP2SAL or PBP3SAL. Affinity of PBP2SAL and PBP3SAL for beta-lactam antibiotics was tested. Relapse after ceftriaxone therapy was analysed in the murine typhoid model. FINDINGS: S. Typhimurium does not express PBP2SAL or PBP3SAL in the Mueller-Hinton medium used for susceptibility testing. The pathogen produces these PBPs in response to acidic pH and nutrient limitation, conditions found in phagosomes of mammalian cells. PBP3SAL has low affinity for beta-lactams, even at acidic pH. In vitro susceptibility to ceftriaxone at low pH is strongly reduced. S. Typhimurium lacking PBP3SAL was unable to cause relapse in mice following ceftriaxone therapy. INTERPRETATION: The reduced capacity of ceftriaxone to clear S. Typhimurium in vivo is favoured by a switch in beta-lactam targets. This switch, involving production of the less-susceptible PBP3SAL, remains invisible for standard procedures used in clinical therapy. We conclude that eradication of salmonellosis will be possible only upon targeting of PBP3SAL with novel drugs.