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 PBP2 SAL or PBP3 SAL . Affinity of PBP2 SAL and PBP3 SAL for beta-lactam antibiotics was tested. Relapse after ceftriaxone therapy was analysed in the murine typhoid model. Findings: S. Typhimurium does not express PBP2 SAL or PBP3 SAL 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. PBP3 SAL has low affinity for beta-lactams, even at acidic pH. In vitro susceptibility to ceftriaxone at low pH is strongly reduced. S. Typhimurium lacking PBP3 SAL 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 PBP3 SAL , remains invisible for standard procedures used in clinical therapy. We conclude that eradication of salmonellosis will be possible only upon targeting of PBP3 SAL with novel drugs.
The peptidoglycan (PG), also known as murein sacculus, is the main component of the bacterial cell wall (Egan et al., 2020). Among its unique physicochemical properties are the presence of D-amino acids and its assembly as a giant covalently bound macromolecule covering the entire cell surface. Because of these properties, the PG determines cell shape and ensures cellular integrity (Egan & Vollmer, 2013;Egan et al., 2017). PG metabolism and morphogenesis have been extensively studied inEscherichia coli and Bacillus subtilis, in which the elongation and division phases are clearly defined in time and space (den Blaauwen et al., 2008;Errington, 2015;Rohs & Bernhardt, 2021). These two phases are orchestrated by PG synthases that crosslink stem peptides in parallel glycan chains (den Blaauwen et al., 2017;Szwedziak & Lowe, 2013). In
Essential peptidoglycan synthases, like penicillin binding proteins 2 and 3 (PBP2/PBP3) of Escherichia coli, define shape by orchestrating cell elongation and division, respectively. Despite being intensively studied as drug targets, the regulatory rules governing their production remain poorly understood. During infection, the closely related pathogen Salmonella enterica serovar Typhimurium downregulates PBP2/PBP3 production and replace them with alternative peptidoglycan synthases, PBP2SAL/PBP3SAL, absent in E. coli. The bases for such switch in morphogenetic proteins are unknown. Here, we show that the S. Typhimurium regulator OmpR triggers PBP2SAL and PBP3SAL expression responding solely to acid pH and define a shared motif present in upstream regions of the PBP2SAL- and PBP3SAL-coding genes sufficient for such control. The elimination of PBP2/PBP3 in infection conditions is however multifactorial, requiring acidity, high osmolarity and being favoured by OmpR and the Prc protease. Remarkably, we found that E. coli loses the essential PBP3 required for cell division when exposed to both acidity and high osmolarity, the environmental cues encountered by intracellular S. Typhimurium. Therefore, OmpR played a central role in the evolution of this pathogen when co-opting the regulation of PBP2SAL/PBP3SAL and, consequently, promoting a new morphogenetic cycle that made possible increasing progeny inside acidic eukaryotic phagosomes.SignificanceSome enzymes that participate in peptidoglycan metabolism are present exclusively in bacterial pathogens and modify its structure to limit immune recognition. The intracellular pathogen Salmonella enterica serovar Typhimurium is the only example known to date in which a “substitution” of essential peptidoglycan enzymes involved in cell division and elongation takes place during infection. The data presented here support instability of PBP3 in environments with acidity and high osmolarity as a probable selective pressure that promoted the fixation of alternative morphogenetic enzymes. This was possible due to the control that OmpR exerted over these new foreign functions. The acquisition of enzymes like PBP2SAL and PBP3SAL therefore represent a “quantum leap” evolutionary event in S. Typhimurium that made possible the colonization of acidic intracellular niches.
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