Biology subjects

Garcia-del Portillo, F.

Publications and source records attributed to Garcia-del Portillo, F..

4 recordsLinked to original sources

bioRxiv2022

Evolutionary analysis in Enterobacterales of the Rcs-repressor protein IgaA unveils two cytoplasmic small β-barrel domains central for function

The Rcs sensor system, comprised by the proteins RcsB/RcsC/RcsD and RcsF, is used by bacteria of the order Enterobacterales to withstand envelope damage. Under non-stress conditions, the system is repressed by the membrane protein IgaA. How IgaA has evolved within Enterobacterales in concert with the Rcs system has not been explored. Here, we report phylogenetic data supporting co-evolution of IgaA with the inner membrane proteins RcsC and RcsD. Functional assays showed that IgaA from representative genera as Shigella and Dickeya, but not those from Yersinia or the endosymbionts Photorhabdus and Sodalis, repress the Rcs system when expressed in a heterogenous host like Salmonella enterica serovar Typhimurium. IgaA structural features have therefore diverged among Enterobacterales. Modelling of IgaA structure unveiled one periplasmic and two cytoplasmic {beta}-rich architectures forming partially-closed small {beta}-barrel (SBB) domains related to OB (oligonucleotide/oligosaccharide binding motif) fold domains. Interactions among conserved residues were mapped in a connector linking SBB-1 domain of cytoplasmic region cyt1 to SBB-2 domain of region cyt2 (residues E180-R265); the C-terminus of cyt1 facing cyt2 (R188-E194-D309 and T191-H326); and, between cyt2-cyt3 regions (H293-E328-R686). These interactions identify a previously unnoticed "hybrid" SBB-2 domain. We also identified interactions absent in the IgaA variants not functional in S. Typhimurium, including H192-P249, which links cyt1 to cyt2, R255-D313 and D287-R314. A short -helix (6) located in the SSB-1 domain is also missing in the non-complementing IgaA tested. Taken together, our data support a central role of the two cytoplasmic SBB domains in IgaA function and evolution. SIGNIFICANCEThe "intracellular growth attenuator A" protein (IgaA) was first reported as repressor of the Rcs system in S. enterica serovar Typhimurium. IgaA orthologs were later studied in other genera and families of the Enterobacterales order, mainly in Escherichia coli. Despite intense investigation about the mechanism by which IgaA controls the Rcs system, the extent at which IgaA evolved within families of the Enterobacterales order has not been investigated. Using a combination of functional assays and in silico structural analyses, our work provides a detail map of conserved and divergent residues in IgaA representing interactions occurring in all Enterobacterales and others that may have diverged concomitantly to interacting proteins, probably for responding to specific environments. Future studies involving mutagenesis of these residues in IgaA of Enterobacterales families and genera of interest will certainly provide valuable insights into the regulation acting in the IgaA-Rcs axis.

microbiology↗

bioRxiv2021

Acid stress signals are integrated into the σB-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes

The general stress response (GSR) in Listeria monocytogenes plays a critical role in the survival of this pathogen in the host gastrointestinal tract. The GSR is regulated by the alternative sigma factor B ({sigma}B), whose role in protection against acid stress is well established. However, the mechanisms leading to its activation by low pH are unknown. Here, we investigated the involvement of the stressosome, a sensory organelle, in transducing low pH signals to induce the GSR. Mild acid shock (15 min at pH 5.0) activated {sigma}B and conferred protection against a subsequent lethal pH challenge. A mutant strain where the stressosome subunit RsbR1 was present but its remaining paralogues were genetically inactivated retained the ability to induce {sigma}B activity at pH 5.0. The role of stressosome phosphorylation in signal transduction was investigated by mutating the putative phosphorylation sites in the core stressosome proteins RsbR1 (rsbR1 T175A, T209A, T241A) and RsbS (rsbS S56A), or in the active site of the stressosome kinase RsbT (rsbT N49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1 T175A variant, retained a near-wild type phenotype. The rsbR1 T209A and rsbR1 T241A mutants displayed constitutive {sigma}B activity. Mild acid shock upregulates invasion genes and stimulates epithelial cell invasion, effects that were abolished in mutants with an inactive or overactive stressosome. Overall, the results show that the stressosome is required for acid-induced activation of {sigma}B in L. monocytogenes. Furthermore, RsbR1 can function independently of its paralogues and that signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209. These insights shed light on the mechanisms of signal transduction that activate the GSR in L. monocytogenes in response to acidic environments, and highlight the role this sensory process in the early stages of the infectious cycle. Author summaryThe stress sensing organelle known as the stressosome, found in many bacterial and archaeal lineages, plays a crucial role in both stress tolerance and virulence in the food-borne pathogen Listeria monocytogenes. However, the mechanisms that lead to its activation and the subsequent activation of the general stress response have remained elusive. In this study, we examined the signal transduction mechanisms that operate in the stressosome in response to acid stress. We found that only one of the five putative sensory proteins present in L. monocytogenes, RsbR1, was required for effective transduction of acid tress signals. We further found that phosphorylation of RsbS and RsbR1, mediated by the RsbT kinase, is essential for signal transduction. Failure to phosphorylate RsbS on Serine 56 completely abolished acid sensing by the stressosome, which prevented the development of adaptive acid tolerance. The acid-induced activation of internalin gene expression was also abolished in mutants with defective stressosome signalling, suggesting a role for the stressosome in the invasion of host cells. Together the data provide new insights into the mechanisms that activate the stressosome in response to acid stress and highlight the role this sensory organelle plays in virulence.

molecular biology↗

bioRxiv2021

Salmonella exploits OmpR to switch essential morphogenetic peptidoglycan synthases in response to intracellular cues

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.

microbiology↗

bioRxiv2020

A secreted NlpC/P60 endopeptidase from Photobacterium damselae subsp. piscicida cleaves the peptidoglycan of potentially competing bacteria

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. Here, it is described the structural and functional characterization of an NlpC/P60-containing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA (Photobacterium NlpC-like Protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the {gamma}-D-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp and neither PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and V. vulnificus. This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA, showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. IMPORTANCEPeptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, originating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or joint its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either for gaining competitive advantages and/or to get nutrients. The specificity of PnpA to the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.

microbiology↗