Javier F. Mariscotti

Modulation of Horizontally Acquired Genes by the Hha-YdgT Proteins in Salmonella enterica Serovar Typhimurium

We describe a transcriptomic study of the effect of hha and ydgT mutations in Salmonella enterica serovar Typhimurium. A large number of genes showing altered expression are located in AT-rich horizontally acquired DNA sequences. Many of these genes have also been reported to be targets for H-NS. As Hha and YdgT interact with H-NS, our findings strongly suggest that Hha and/or YdgT must form complexes with H-NS when they silence these DNA regions.

Genome Expression Analyses Revealing the Modulation of the Salmonella Rcs Regulon by the Attenuator IgaA

Intracellular growth attenuator A (IgaA) was identified as a Salmonella enterica regulator limiting bacterial growth inside fibroblasts. Genetic evidence further linked IgaA to repression of the RcsCDB regulatory system, which responds to envelope stress. How IgaA attenuates this system is unknown. Here, we present genome expression profiling data of S. enterica serovar Typhimurium igaA mutants grown at high osmolarity and displaying exacerbated Rcs responses. Transcriptome data revealed that IgaA attenuates gene expression changes requiring phosphorylated RcsB (RcsB~P) activity. Some RcsB-regulated genes, yciGFE and STM1862 (pagO)-STM1863-STM1864, were equally expressed in wild-type and igaA strains, suggesting a maximal expression at low levels of RcsB ~P. Other genes, such as metB, ypeC, ygaC, glnK, glnP, napA, glpA, and nirB, were shown for the first time and by independent methods to be regulated by the RcsCDB system. Interestingly, IgaA-deficient strains with reduced RcsC or RcsD levels exhibited different Rcs responses and distinct virulence properties. spv virulence genes were differentially expressed in most of the analyzed strains. spvA expression required RcsB and IgaA but, unexpectedly, was also impaired upon stimulation of the RcsC-->RcsD-->RcsB phosphorelay. Overproduction of either RcsB(+) or a nonphosphorylatable RcsB(D56Q) variant in strains displaying low spvA expression unveiled that both dephosphorylated RcsB and RcsB~P are required for optimal spvA expression. Taken together, our data support a model with IgaA attenuating the RcsCDB system by favoring the switch of RcsB~P to the dephosphorylated state. This role of IgaA in constantly fine-tuning the RcsB~P/RcsB ratio may ensure the proper expression of important virulence factors, such as the Spv proteins.

The Listeria monocytogenes Sortase-B Recognizes Varied Amino Acids at Position 2 of the Sorting Motif

Sortases are bacterial enzymes that anchor surface proteins covalently to the peptidoglycan upon cleavage of a motif located at their C-terminal end. Motifs recognized by sortases of the class-B (SrtB) are defined by the consensus sequence NP(Q/K)(T/S)(N/G/S)(D/A). Evidence supporting this consensus is limited to IsdC of Staphylococcus aureus and Bacillus anthracis, cleaved at motifs NPQTN and NPKTG, respectively. In Listeria monocytogenes, StrB has two substrates, Lmo2185 and Lmo2186, containing NAKTN and NKVTN (or the overlapping sequence NPKSS) as putative sorting motifs. Some of these motifs do not match the consensus, because they lack either proline (P) at position 2 or glutamine/lysine (Q/K) at position 3. Here, we identified NPKSS as a sorting motif of Lmo2186 by monitoring anchoring to peptidoglycan of chimeras lacking each of its two predicted motifs. Motif-swapping experiments confirmed that NPKSS, but not NKVTN, could replace NAKTN for anchoring of an Lmo2185 chimera. Residue substitutions in the NPKSS sequence revealed the essentiality of proline at position 2 for recognition of this particular motif. Lysine at position 3 was however dispensable. Deletion of NAKTN, on the other hand, abrogated SrtB-mediated anchoring of the Lmo2185 chimera. NAKTN, therefore, represents an exception to the rule of a conserved proline in position 2 of the sorting motif. Taken together, our data indicate that proline is not absolutely required for substrate recognition by sortases of the class-B. In addition, they prove the capacity of a single sortase, as SrtB of L. monocytogenes, to recognize varied amino acids at position 2 of the sorting motif.

Glycine Betaine Transmethylase Mutant of Pseudomonas aeruginosa

The gene for glycine betaine transmethylase (gbt) was identified in Pseudomonas aeruginosa strain Fildes III by biochemical, physiological, and molecular approaches. Based on sequence analysis, the knockout gene corresponded to an open reading frame (ORF) named PA3082 in the genome of P. aeruginosa PAO1. The translated product of this ORF displayed similarity to transferases of different microorganisms. Mutation in gbt blocked the utilization of choline and glycine betaine as carbon and nitrogen sources.

The Listeria monocytogenes LPXTG surface protein Lmo1413 is an invasin with capacity to bind mucin

Many Gram-positive bacterial pathogens use surface proteins covalently anchored to the peptidoglycan to cause disease. Bacteria of the genus Listeria have the largest number of surface proteins of this family. Every Listeria genome sequenced to date contains more than forty genes encoding surface proteins bearing anchoring-domains with an LPXTG motif that is recognized for covalent linkage to the peptidoglycan. About one-third of these proteins are present exclusively in pathogenic Listeria species, with some of them acting as adhesins or invasins that promote bacterial entry into eukaryotic cells. Here, we investigated two LPXTG surface proteins of the pathogen L. monocytogenes, Lmo1413 and Lmo2085, of unknown function and absent in non-pathogenic Listeria species. Lack of these two proteins does not affect bacterial adhesion or invasion of host cells using in vitro infection models. However, expression of Lmo1413 promotes entry of the non-invasive species L. innocua into non-phagocytic host cells, an effect not observed with Lmo2085. Moreover, overproduction of Lmo1413, but not Lmo2085, increases the invasion rate in non-phagocytic eukaryotic cells of an L. monocytogenes mutant deficient in the acting-binding protein ActA. Unexpectedly, production of full-length Lmo1413 and InlA exhibited opposite trends in a high percentage of L. monocytogenes isolates obtained from different sources. The idea of Lmo1413 playing a role as a new auxiliary invasin was also sustained by assays revealing that purified Lmo1413 binds to mucin via its MucBP domains. Taken together, these data indicate that Lmo1413, which we rename LmiA, for Listeria-mucin-binding invasin-A, may promote interaction of bacteria with adhesive host protective components and, in this manner, facilitate bacterial entry.

Inverse regulation in the metabolic genes pckA and metE revealed by proteomic analysis of the Salmonella RcsCDB regulon.

The RcsC, RcsD, and RcsB proteins compose a system used by enteric bacteria to sense envelope stress. Signal transmission occurs from the sensor RcsC to the transcriptional regulator RcsB. Accessory proteins, such as IgaA, are known to adjust the response level. In a previous transcriptomic study, we uncovered 85 genes differentially expressed in Salmonella enterica serovar Typhimurium igaA mutants. Here, we extended these observations to proteomics by performing differential isotope-coded protein labeling (ICPL) followed by liquid chromatography-electrospray ionization tandem mass spectrometry. Five-hundred five proteins were identified and quantified, with 75 of them displaying significant changes in response to alterations in the RcsCDB system. Divergent expression at the RNA and protein level was observed for the metabolic genes pckA and metE, involved in gluconeogenesis and methionine synthesis, respectively. When analyzed in diverse environmental conditions, including the intracellular niche of eukaryotic cells, inverse regulation was more evident for metE and in bacteria growing in defined minimal medium or to stationary phase. The RcsCDB system was also shown to repress the synthesis of the small RNA FnrS, previously reported to modulate metE expression. Collectively, these findings provide new insights into post-transcriptional regulatory mechanisms involving the RcsCDB system and its control over metabolic functions.

Occurrence of mutations impairing sigma factor B (SigB) function upon inactivation of Listeria monocytogenes genes encoding surface proteins.

Bacteria of the genus Listeria contain the largest family of LPXTG surface proteins covalently anchored to the peptidoglycan. The extent to which these proteins may function or be regulated cooperatively is at present unknown. Because of their unique cellular location, we reasoned that distinct LPXTG proteins could act as elements contributing to cell wall homeostasis or influencing the stability of other surface proteins bound to peptidoglycan. To test this hypothesis, we used proteomics to analyse mutants of the intracellular pathogen Listeria monocytogenes lacking distinct LPXTG proteins implicated in pathogen-host interactions, such as InlA, InlF, InlG, InlH, InlJ, LapB and Vip. Changes in the cell wall proteome were found in inlG and vip mutants, which exhibited reduced levels of the LPXTG proteins InlH, Lmo0610, Lmo0880 and Lmo2085, all regulated by the stress-related sigma factor SigB. The ultimate basis of this alteration was uncovered by genome sequencing, which revealed that these inlG and vip mutants carried loss-of-function mutations in the rsbS, rsbU and rsbV genes encoding regulatory proteins that control SigB activity. Attempts to recapitulate this negative selection of SigB in a large series of new inlG or vip mutants constructed for this purpose were, however, unsuccessful. These results indicate that inadvertent secondary mutations affecting SigB functionality can randomly arise in L. monocytogenes when using common genetic procedures or during subculturing. Testing of SigB activity could be therefore valuable when manipulating genetically L. monocytogenes prior to any subsequent phenotypic analysis. This test may be even more justified when generating deletions affecting cell envelope components.

CpxR-Dependent Thermoregulation of Serratia marcescens PrtA Metalloprotease Expression and Its Contribution to Bacterial Biofilm Formation

PrtA is the major secreted metalloprotease of Serratia marcescens Previous reports implicate PrtA in the pathogenic capacity of this bacterium. PrtA is also clinically used as a potent analgesic and anti-inflammatory drug, and its catalytic properties attract industrial interest. Comparatively, there is scarce knowledge about the mechanisms that physiologically govern PrtA expression in Serratia In this work, we demonstrate that PrtA production is derepressed when the bacterial growth temperature decreases from 37°C to 30°C. We show that this thermoregulation occurs at the transcriptional level. We determined that upstream of prtA, there is a conserved motif that is directly recognized by the CpxR transcriptional regulator. This feature is found along Serratia strains irrespective of their isolation source, suggesting an evolutionary conservation of CpxR-dependent regulation of PrtA expression. We found that in S. marcescens, the CpxAR system is more active at 37°C than at 30°C. In good agreement with these results, in a cpxR mutant background, prtA is derepressed at 37°C, while overexpression of the NlpE lipoprotein, a well-known CpxAR-inducing condition, inhibits PrtA expression, suggesting that the levels of the activated form of CpxR are increased at 37°C over those at 30°C. In addition, we establish that PrtA is involved in the ability of S. marcescens to develop biofilm. In accordance, CpxR influences the biofilm phenotype only when bacteria are grown at 37°C. In sum, our findings shed light on regulatory mechanisms that fine-tune PrtA expression and reveal a novel role for PrtA in the lifestyle of S. marcescensIMPORTANCE We demonstrate that S. marcescens metalloprotease PrtA expression is transcriptionally thermoregulated. While strongly activated below 30°C, its expression is downregulated at 37°C. We found that in S. marcescens, the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is activated at 37°C and able to downregulate PrtA expression by direct interaction of CpxR with a binding motif located upstream of the prtA gene. Moreover, we reveal that PrtA expression favors the ability of S. marcescens to develop biofilm, irrespective of the bacterial growth temperature. In this context, thermoregulation along with a highly conserved CpxR-dependent modulation mechanism gives clues about the relevance of PrtA as a factor implicated in the persistence of S. marcescens on abiotic surfaces and in bacterial host colonization capacity.