scholarly journals Roles of the Outer Membrane Protein AsmA of Salmonella enterica in the Control of marRAB Expression and Invasion of Epithelial Cells

2009 ◽  
Vol 191 (11) ◽  
pp. 3615-3622 ◽  
Author(s):  
Ana I. Prieto ◽  
Sara B. Hernández ◽  
Ignacio Cota ◽  
M. Graciela Pucciarelli ◽  
Yuri Orlov ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Transmembrane Domain ◽  
In Silico Analysis ◽  
Sodium Deoxycholate ◽  
Oral Route ◽  
Serovar Typhimurium ◽  
Bile Resistance

ABSTRACT A genetic screen for suppressors of bile sensitivity in DNA adenine methylase (dam) mutants of Salmonella enterica serovar Typhimurium yielded insertions in an uncharacterized locus homologous to the Escherichia coli asmA gene. Disruption of asmA suppressed bile sensitivity also in phoP and wec mutants of S. enterica and increased the MIC of sodium deoxycholate for the parental strain ATCC 14028. Increased levels of marA mRNA were found in asmA, asmA dam, asmA phoP, and asmA wec strains of S. enterica, suggesting that lack of AsmA activates expression of the marRAB operon. Hence, asmA mutations may enhance bile resistance by inducing gene expression changes in the marRAB-controlled Mar regulon. In silico analysis of AsmA structure predicted the existence of one transmembrane domain. Biochemical analysis of subcellular fractions revealed that the asmA gene of S. enterica encodes a protein of ∼70 kDa located in the outer membrane. Because AsmA is unrelated to known transport and/or efflux systems, we propose that activation of marRAB in asmA mutants may be a consequence of envelope reorganization. Competitive infection of BALB/c mice with asmA + and asmA isogenic strains indicated that lack of AsmA attenuates Salmonella virulence by the oral route but not by the intraperitoneal route. Furthermore, asmA mutants showed a reduced ability to invade epithelial cells in vitro.

scholarly journals Association of a Protective Monoclonal IgA with the O Antigen of Salmonella enterica Serovar Typhimurium Impacts Type 3 Secretion and Outer Membrane Integrity

2012 ◽  
Vol 80 (7) ◽  
pp. 2454-2463 ◽  
Author(s):  
Stephen J. Forbes ◽  
Daniel Martinelli ◽  
Chyongere Hsieh ◽  
Jeffrey G. Ault ◽  
Michael Marko ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Epithelial Cells ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Integrity ◽  
Effector Proteins ◽  
Content Type ◽  
O Antigen ◽  
Serovar Typhimurium ◽  
Type 3

ABSTRACTInvasion of intestinal epithelial cells bySalmonella entericaserovar Typhimurium is an energetically demanding process, involving the transfer of effector proteins from invading bacteria into host cells via a specialized organelle known as theSalmonellapathogenicity island 1 (SPI-1) type 3 secretion system (T3SS). By a mechanism that remains poorly understood, entry ofS. Typhimurium into epithelial cells is inhibited by Sal4, a monoclonal, polymeric IgA antibody that binds an immunodominant epitope within the O-antigen (O-Ag) component of lipopolysaccharide. In this study, we investigated how the binding of Sal4 to the surface ofS. Typhimurium influences T3SS activity, bacterial energetics, and outer membrane integrity. We found that Sal4 treatment impaired T3SS-mediated translocon formation and attenuated the delivery of tagged effector proteins into epithelial cells. Sal4 treatment coincided with a partial reduction in membrane energetics and intracellular ATP levels, possibly explaining the impairment in T3SS activity. Sal4's effects on bacterial secretion and energetics occurred concurrently with an increase in O-Ag levels in culture supernatants, alterations in outer membrane permeability, and changes in surface ultrastructure, as revealed by transmission electron microscopy and cryo-electron microscopy. We propose that Sal4, by virtue of its ability to bind and cross-link the O-Ag, induces a form of outer membrane stress that compromises the integrity of theS. Typhimurium cell envelope and temporarily renders the bacterium avirulent.

scholarly journals QseC Mediates Salmonella enterica Serovar Typhimurium Virulence In Vitro and In Vivo

2009 ◽  
Vol 78 (3) ◽  
pp. 914-926 ◽  
Author(s):  
Cristiano G. Moreira ◽  
David Weinshenker ◽  
Vanessa Sperandio
Keyword(s):  
Epithelial Cells ◽  
Salmonella Enterica ◽  
Systemic Disease ◽  
Systemic Infection ◽  
Flagellar Motility ◽  
Signaling System ◽  
Interkingdom Signaling ◽  
Serovar Typhimurium

ABSTRACT The autoinducer-3 (AI-3)/epinephrine (Epi)/norepinephrine (NE) interkingdom signaling system mediates chemical communication between bacteria and their mammalian hosts. The three signals are sensed by the QseC histidine kinase (HK) sensor. Salmonella enterica serovar Typhimurium is a pathogen that uses HKs to sense its environment and regulate virulence. Salmonella serovar Typhimurium invades epithelial cells and survives within macrophages. Invasion of epithelial cells is mediated by the type III secretion system (T3SS) encoded in Salmonella pathogenicity island 1 (SPI-1), while macrophage survival and systemic disease are mediated by the T3SS encoded in SPI-2. Here we show that QseC plays an important role in Salmonella serovar Typhimurium pathogenicity. A qseC mutant was impaired in flagellar motility, in invasion of epithelial cells, and in survival within macrophages and was attenuated for systemic infection in 129x1/SvJ mice. QseC acts globally, regulating expression of genes within SPI-1 and SPI-2 in vitro and in vivo (during infection of mice). Additionally, dopamine β-hydroxylase knockout (Dbh − / −) mice that do not produce Epi or NE showed different susceptibility to Salmonella serovar Typhimurium infection than wild-type mice. These data suggest that the AI-3/Epi/NE signaling system is a key factor during Salmonella serovar Typhimurium pathogenesis in vitro and in vivo. Elucidation of the role of this interkingdom signaling system in Salmonella serovar Typhimurium should contribute to a better understanding of the complex interplay between the pathogen and the host during infection.

scholarly journals Interplay between the QseC and QseE Bacterial Adrenergic Sensor Kinases in Salmonella enterica Serovar Typhimurium Pathogenesis

2012 ◽  
Vol 80 (12) ◽  
pp. 4344-4353 ◽  
Author(s):  
Cristiano G. Moreira ◽  
Vanessa Sperandio
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Salmonella Enterica ◽  
Double Mutant ◽  
Virulence Gene ◽  
Virulence Gene Expression ◽  
Regulatory Cascade ◽  
Serovar Typhimurium ◽  
Sensor Kinases

ABSTRACTThe bacterial adrenergic sensor kinases QseC and QseE respond to epinephrine and/or norepinephrine to initiate a complex phosphorelay regulatory cascade that modulates virulence gene expression in several pathogens. We have previously shown that QseC activates virulence gene expression inSalmonella entericaserovar Typhimurium. Here we report the role of QseE inS. Typhimurium pathogenesis as well as the interplay between these two histidine sensor kinases in gene regulation. AnS. TyphimuriumqseEmutant is hampered in the invasion of epithelial cells and intramacrophage replication. The ΔqseCstrain is highly attenuated for intramacrophage survival but has only a minor defect in invasion. However, the ΔqseECstrain has only a slight attenuation in invasion, mirroring the ΔqseCstrain, and has an intermediary intramacrophage replication defect in comparison to the ΔqseEand ΔqseCstrains. The expressions of thesipAandsopBgenes, involved in the invasion of epithelial cells, are activated by epinephrine via QseE. The expression levels of these genes are still decreased in the ΔqseECdouble mutant, albeit to a lesser extent, congruent with the invasion phenotype of this mutant. The expression level of thesifAgene, important for intramacrophage replication, is decreased in theqseEmutant and the ΔqseECdouble mutant grownin vitro. However, as previously reported by us, the epinephrine-dependent activation of this gene occurs via QseC. In the systemic model ofS. Typhimurium infection of BALB/c mice, theqseCandqseEmutants are highly attenuated, while the double mutant has an intermediary phenotype. Altogether, these data suggest that both adrenergic sensors play an important role in modulating several aspects ofS. Typhimurium pathogenesis.

scholarly journals Flagellar Phase Variation of Salmonella enterica Serovar Typhimurium Contributes to Virulence in the Murine Typhoid Infection Model but Does Not InfluenceSalmonella-Induced Enteropathogenesis

2001 ◽  
Vol 69 (5) ◽  
pp. 3021-3030 ◽  
Author(s):  
Jack S. Ikeda ◽  
Clare K. Schmitt ◽  
Stephen C. Darnell ◽  
Patricia R. Watson ◽  
Jennifer Bispham ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Salmonella Enterica ◽  
Phase Variation ◽  
Selective Advantage ◽  
Peritoneal Macrophages ◽  
Infection Model ◽  
Wild Type ◽  
Two Phase ◽  
Serovar Typhimurium

ABSTRACT Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC+phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.

scholarly journals Outer Membrane Structural Defects in Salmonella enterica Serovar Typhimurium Affect Neutrophil Chemokinesis but Not Chemotaxis

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Eric J. Leaman ◽  
Alvin Aung ◽  
Alexie J. Jacques ◽  
Bahareh Behkam
Keyword(s):  
Steady State ◽  
Single Cell ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Structural Defects ◽  
Serovar Typhimurium ◽  
Microbial Strains ◽  
Neutrophil Response ◽  
Transient And Steady State

ABSTRACT Neutrophils, the first line of defense against pathogens, are critical in the host response to acute and chronic infections. In Gram-negative pathogens, the bacterial outer membrane (OM) is a key mediator of pathogen detection; nonetheless, the effects of variations in its molecular structure on the neutrophil migratory response to bacteria remain largely unknown. Here, we developed a quantitative microfluidic assay that precludes physical contact between bacteria and neutrophils while maintaining chemical communication, thus allowing investigation of both transient and steady-state responses of neutrophils to a library of Salmonella enterica serovar Typhimurium OM-related mutants at single-cell resolution. Using single-cell quantitative metrics, we found that transient neutrophil chemokinesis is highly gradated based upon OM structure, while transient and steady-state chemotaxis responses differ little between mutants. Based on our finding of a lack of correlation between chemokinesis and chemotaxis, we define “stimulation score” as a metric that comprehensively describes the neutrophil response to pathogens. Complemented with a killing assay, our results provide insight into how OM modifications affect neutrophil recruitment and pathogen survival. Altogether, our platform enables the discovery of transient and steady-state migratory responses and provides a new path for quantitative interrogation of cell decision-making processes in a variety of host-pathogen interactions. IMPORTANCE Our findings provide insights into the previously unexplored effects of Salmonella envelope defects on fundamental innate immune cell behavior, which advance the knowledge in pathogen-host cell biology and potentially inspire the rational design of attenuated strains for vaccines or immunotherapeutic strains for cancer therapy. Furthermore, the microfluidic assay platform and analytical tools reported herein enable high-throughput, sensitive, and quantitative screening of microbial strains' immunogenicity in vitro. This approach could be particularly beneficial for rapid in vitro screening of engineered microbial strains (e.g., vaccine candidates) as the quantitative ranking of the overall strength of the neutrophil response, reported by “stimulation score,” agrees with in vivo cytokine response trends reported in the literature.

scholarly journals Phosphate Groups of Lipid A Are Essential for Salmonella enterica Serovar Typhimurium Virulence and Affect Innate and Adaptive Immunity

2012 ◽  
Vol 80 (9) ◽  
pp. 3215-3224 ◽  
Author(s):  
Qingke Kong ◽  
David A. Six ◽  
Qing Liu ◽  
Lillian Gu ◽  
Shifeng Wang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Adaptive Immunity ◽  
Salmonella Enterica ◽  
Lipid A ◽  
Outer Membrane Proteins ◽  
Polymyxin B ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Phosphate Groups

ABSTRACTLipid A is a key component of the outer membrane of Gram-negative bacteria and stimulates proinflammatory responses via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. Its endotoxic activity depends on the number and length of acyl chains and its phosphorylation state. InSalmonella entericaserovar Typhimurium, removal of the secondary laurate or myristate chain in lipid A results in bacterial attenuation and growth defectsin vitro. However, the roles of the two lipid A phosphate groups in bacterial virulence and immunogenicity remain unknown. Here, we used anS. TyphimuriummsbB pagL pagP lpxRmutant, carrying penta-acylated lipid A, as the parent strain to construct a series of mutants synthesizing 1-dephosphorylated, 4′-dephosphorylated, or nonphosphorylated penta-acylated lipid A. Dephosphorylated mutants exhibited increased sensitivity to deoxycholate and showed increased resistance to polymyxin B. Removal of both phosphate groups severely attenuated the mutants when administered orally to BALB/c mice, but the mutants colonized the lymphatic tissues and were sufficiently immunogenic to protect the host from challenge with wild-typeS. Typhimurium. Mice receivingS. Typhimurium with 1-dephosphorylated or nonphosphorylated penta-acylated lipid A exhibited reduced levels of cytokines. Attenuated and dephosphorylatedSalmonellavaccines were able to induce adaptive immunity against heterologous (PspA ofStreptococcus pneumoniae) and homologous antigens (lipopolysaccharide [LPS] and outer membrane proteins [OMPs]).

TolA mediates the differential detergent resistance pattern between the Salmonella enterica subsp. enterica serovars Typhi and Typhimurium

Microbiology ◽  
2011 ◽  
Vol 157 (5) ◽  
pp. 1402-1415 ◽  
Author(s):  
Amit Lahiri ◽  
T. K. Ananthalakshmi ◽  
Arvindhan G. Nagarajan ◽  
Seemun Ray ◽  
Dipshikha Chakravortty
Keyword(s):  
Outer Membrane ◽  
Salmonella Enterica ◽  
Single Cells ◽  
Membrane Integrity ◽  
Luria Broth ◽  
Resistance Pattern ◽  
Significant Difference ◽  
Serovar Typhimurium ◽  
Bile Resistance

The tol–pal genes are essential for maintaining the outer membrane integrity and detergent resistance in various Gram-negative bacteria, including Salmonella. The role of TolA has been well established for the bile resistance of Salmonella enterica subsp. enterica serovar Typhimurium. We compared the bile resistance pattern between the S. enterica serovars Typhi and Typhimurium and observed that Typhi is more resistant to bile-mediated damage. A closer look revealed a significant difference in the TolA sequence between the two serovars which contributes to the differential detergent resistance. The tolA knockout of both the serovars behaves completely differently in terms of membrane organization and morphology. The role of the Pal proteins and difference in LPS organization between the two serovars were verified and were found to have no direct connection with the altered bile resistance. In normal Luria broth (LB), S. Typhi ΔtolA is filamentous while S. Typhimurium ΔtolA grows as single cells, similar to the wild-type. In low osmolarity LB, however, S. Typhimurium ΔtolA started chaining and S. Typhi ΔtolA showed no growth. Further investigation revealed that the chaining phenomenon observed was the result of failure of the outer membrane to separate in the dividing cells. Taken together, the results substantiate the evolution of a shorter TolA in S. Typhi to counteract high bile concentrations, at the cost of lower osmotic tolerance.

scholarly journals Protecting against Antimicrobial Effectors in the Phagosome Allows SodCII To Contribute to Virulence in Salmonella enterica Serovar Typhimurium

2010 ◽  
Vol 192 (8) ◽  
pp. 2140-2149 ◽  
Author(s):  
Byoungkwan Kim ◽  
Susan M. Richards ◽  
John S. Gunn ◽  
James M. Slauch
Keyword(s):  
Antimicrobial Peptides ◽  
Antimicrobial Peptide ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Osmotic Shock ◽  
Polymyxin B ◽  
Antimicrobial Substances ◽  
Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium replicates in macrophages, where it is subjected to antimicrobial substances, including superoxide, antimicrobial peptides, and proteases. The bacterium produces two periplasmic superoxide dismutases, SodCI and SodCII. Although both are expressed during infection, only SodCI contributes to virulence in the mouse by combating phagocytic superoxide. The differential contribution to virulence is at least partially due to inherent differences in the SodCI and SodCII proteins that are independent of enzymatic activity. SodCII is protease sensitive, and like other periplasmic proteins, it is released by osmotic shock. In contrast, SodCI is protease resistant and is retained within the periplasm after osmotic shock, a phenomenon that we term “tethering.” We hypothesize that in the macrophage, antimicrobial peptides transiently disrupt the outer membrane. SodCII is released and/or phagocytic proteases gain access to the periplasm, and SodCII is degraded. SodCI is tethered within the periplasm and is protease resistant, thereby remaining to combat superoxide. Here we test aspects of this model. SodCII was released by the antimicrobial peptide polymyxin B or a mouse macrophage antimicrobial peptide (CRAMP), while SodCI remained tethered within the periplasm. A Salmonella pmrA constitutive mutant no longer released SodCII in vitro. Moreover, in the constitutive pmrA background, SodCII could contribute to survival of Salmonella during infection. SodCII also provided a virulence benefit in mice genetically defective in production of CRAMP. Thus, consistent with our model, protecting the outer membrane against antimicrobial peptides allows SodCII to contribute to virulence in vivo. These data also suggest direct in vivo cooperative interactions between macrophage antimicrobial effectors.

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