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 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 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]).

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.

scholarly journals Murein Lipoprotein Is a Critical Outer Membrane Component Involved in Salmonella enterica Serovar Typhimurium Systemic Infection

2005 ◽  
Vol 73 (2) ◽  
pp. 1081-1096 ◽  
Author(s):  
A. A. Fadl ◽  
J. Sha ◽  
G. R. Klimpel ◽  
J. P. Olano ◽  
D. W. Niesel ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Salmonella Enterica ◽  
Lipid A ◽  
Enteric Bacteria ◽  
Host Cells ◽  
Double Knockout ◽  
In Vivo Models ◽  
Serovar Typhimurium

ABSTRACT Lipopolysaccharide (LPS) and Braun (murein) lipoprotein (Lpp) are major components of the outer membrane of gram-negative enteric bacteria that function as potent stimulators of inflammatory and immune responses. In a previous paper, we provided evidence that two functional copies of the lipoprotein gene (lppA and lppB) located on the chromosome of Salmonella enterica serovar Typhimurium contributed to bacterial virulence. In this study, we characterized lppA and lppB single-knockout (SKO) mutants and compared them with an lpp double-knockout (DKO) mutant using in vitro and in vivo models. Compared to the lpp DKO mutant, which was nonmotile, the motility of the lpp SKO mutants was significantly increased (73 to 77%), although the level of motility did not reach the level of wild-type (WT) S. enterica serovar Typhimurium. Likewise, the cytotoxicity was also significantly increased when T84 human intestinal epithelial cells and RAW264.7 murine macrophages were infected with the lpp SKO mutants compared to the cytotoxicity when cells were infected with the lpp DKO mutant. The level of interleukin-8 (IL-8) in polarized T84 cells infected with the lppB SKO mutant was significantly higher (two- to threefold higher), reaching the level in cells infected with WT S. enterica serovar Typhimurium, than the level in host cells infected with the lppA SKO mutant. The lpp DKO mutant induced minimal levels of IL-8. Similarly, sera from mice infected with the lppB SKO mutant contained 4.5- to 10-fold-higher levels of tumor necrosis factor-α and IL-6; the levels of these cytokines were 1.7- to 3.0-fold greater in the lppA SKO mutant-infected mice than in animals challenged with the lpp DKO mutant. The increased cytokine levels observed with the lppB SKO mutant in mice correlated with greater tissue damage in the livers and spleens of these mice than in the organs of animals infected with the lppA SKO and lpp DKO mutants. Moreover, the lppB SKO mutant-infected mice had increased susceptibility to death. Since the lpp DKO mutant retained intact LPS, we constructed an S. enterica serovar Typhimurium triple-knockout (TKO) mutant in which the lppA and lppB genes were deleted from an existing msbB mutant (msbB encodes an enzyme required for the acylation of lipid A). Compared to the lpp DKO and msbB SKO mutants, the lpp-msbB TKO mutant was unable to induce cytotoxicity and to produce cytokines and chemokines in vitro and in vivo. These studies provided the first evidence of the relative contributions of Lpp and lipid A acylation to Salmonella pathogenesis.

Integration host factor alleviates H-NS silencing of the Salmonella enterica serovar Typhimurium master regulator of SPI1, hilA

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2504-2514 ◽  
Author(s):  
Mário H. Queiroz ◽  
Cristina Madrid ◽  
Sònia Paytubi ◽  
Carlos Balsalobre ◽  
Antonio Juárez
Keyword(s):  
Stationary Phase ◽  
Salmonella Enterica ◽  
Growth Conditions ◽  
Integration Host Factor ◽  
Band Shift ◽  
Global Regulators ◽  
Serovar Typhimurium ◽  
Associated Proteins ◽  
Transcription Data

Coordination of the expression of Salmonella enterica invasion genes on Salmonella pathogenicity island 1 (SPI1) depends on a complex circuit involving several regulators that converge on expression of the hilA gene, which encodes a transcriptional activator (HilA) that modulates expression of the SPI1 virulence genes. Two of the global regulators that influence hilA expression are the nucleoid-associated proteins Hha and H-NS. They interact and form a complex that modulates gene expression. A chromosomal transcriptional fusion was constructed to assess the effects of these modulators on hilA transcription under several environmental conditions as well as at different stages of growth. The results obtained showed that these proteins play a role in silencing hilA expression at both low temperature and low osmolarity, irrespective of the growth phase. H-NS accounts for the main repressor activity. At high temperature and osmolarity, H-NS-mediated silencing completely ceases when cells enter the stationary phase, and hilA expression is induced. Mutants lacking IHF did not induce hilA in cells entering the stationary phase, and this lack of induction was dependent on the presence of H-NS. Band-shift assays and in vitro transcription data showed that for hilA induction under certain growth conditions, IHF is required to alleviate H-NS-mediated silencing.

scholarly journals Caveolin-1-Deficient Mice Show Defects in Innate Immunity and Inflammatory Immune Response during Salmonella enterica Serovar Typhimurium Infection

2006 ◽  
Vol 74 (12) ◽  
pp. 6665-6674 ◽  
Author(s):  
Freddy A. Medina ◽  
Cecilia J. de Almeida ◽  
Elliott Dew ◽  
Jiangwei Li ◽  
Gloria Bonuccelli ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Salmonella Enterica ◽  
Inflammatory Responses ◽  
Systemic Infection ◽  
Bacterial Invasion ◽  
Chemokine Production ◽  
Caveolin 1 ◽  
Serovar Typhimurium ◽  
Deficient Mice

ABSTRACT A number of studies have shown an association of pathogens with caveolae. To this date, however, there are no studies showing a role for caveolin-1 in modulating immune responses against pathogens. Interestingly, expression of caveolin-1 has been shown to occur in a regulated manner in immune cells in response to lipopolysaccharide (LPS). Here, we sought to determine the role of caveolin-1 (Cav-1) expression in Salmonella pathogenesis. Cav-1−/− mice displayed a significant decrease in survival when challenged with Salmonella enterica serovar Typhimurium. Spleen and tissue burdens were significantly higher in Cav-1−/− mice. However, infection of Cav-1−/− macrophages with serovar Typhimurium did not result in differences in bacterial invasion. In addition, Cav-1−/− mice displayed increased production of inflammatory cytokines, chemokines, and nitric oxide. Regardless of this, Cav-1−/− mice were unable to control the systemic infection of Salmonella. The increased chemokine production in Cav-1−/− mice resulted in greater infiltration of neutrophils into granulomas but did not alter the number of granulomas present. This was accompanied by increased necrosis in the liver. However, Cav-1−/− macrophages displayed increased inflammatory responses and increased nitric oxide production in vitro in response to Salmonella LPS. These results show that caveolin-1 plays a key role in regulating anti-inflammatory responses in macrophages. Taken together, these data suggest that the increased production of toxic mediators from macrophages lacking caveolin-1 is likely to be responsible for the marked susceptibility of caveolin-1-deficient mice to S. enterica serovar Typhimurium.

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 Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines againstStreptococcus pneumoniaeandSalmonella entericaSerovar Typhi

2018 ◽  
Vol 86 (9) ◽  
Author(s):  
Vivek Belde ◽  
Matthew P. Cravens ◽  
Dania Gulandijany ◽  
Justin A. Walker ◽  
Isabel Palomo-Caturla ◽  
...  
Keyword(s):  
Antibody Response ◽  
B Cell ◽  
Salmonella Enterica ◽  
Passive Immunization ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Human Infants ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTB cell antigen receptor (BCR) diversity increases by several orders of magnitude due to the action of terminal deoxynucleotidyl transferase (TdT) during V(D)J recombination. Unlike adults, infants have limited BCR diversity, in part due to reduced expression of TdT. Since human infants and young mice respond poorly to polysaccharide vaccines, such as the pneumococcal polysaccharide vaccine Pneumovax23 and Vi polysaccharide (ViPS) ofSalmonella entericaserovar Typhi, we tested the contribution of TdT-mediated BCR diversity in response to these vaccines. We found that TdT+/−and TdT−/−mice generated comparable antibody responses to Pneumovax23 and survivedStreptococcus pneumoniaechallenge. Moreover, passive immunization of B cell-deficient mice with serum from Pneumovax23-immunized TdT+/−or TdT−/−mice conferred protection. TdT+/−and TdT−/−mice generated comparable levels of anti-ViPS antibodies and antibody-dependent, complement-mediated bactericidal activity againstS. Typhiin vitro. To test the protective immunity conferred by ViPS immunizationin vivo, TdT+/−and TdT−/−mice were challenged with a chimericSalmonella entericaserovar Typhimurium strain expressing ViPS, since mice are nonpermissive hosts forS. Typhi infection. Compared to their unimmunized counterparts, immunized TdT+/−and TdT−/−mice challenged with ViPS-expressingS. Typhimurium exhibited a significant reduction in the bacterial burden and liver pathology. These data suggest that the impaired antibody response to the Pneumovax23 and ViPS vaccines in the young is not due to limited TdT-mediated BCR diversification.

scholarly journals Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles by Using Protein Ligation

2018 ◽  
Vol 84 (8) ◽  
pp. e02567-17 ◽  
Author(s):  
H. Bart van den Berg van Saparoea ◽  
Diane Houben ◽  
Marien I. de Jonge ◽  
Wouter S. P. Jong ◽  
Joen Luirink
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Vesicles ◽  
Therapeutic Agents ◽  
Outer Membrane Vesicles ◽  
High Density ◽  
Content Type ◽  
Protein Ligation ◽  
Serovar Typhimurium

ABSTRACT The Escherichia coli virulence factor hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on live E. coli and Salmonella enterica serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences can be genetically fused into the Hbp core structure for optimal exposure to the immune system. Although the Hbp display platform is relatively tolerant, increasing the number, size, and complexity of integrated sequences generally lowers the expression of the fused constructs and limits the density of display. This is due to the intricate mechanism of Hbp secretion across the outer membrane and the efficient quality control of translocation-incompetent chimeric Hbp molecules in the periplasm. To address this shortcoming, we explored the coupling of purified proteins to the Hbp carrier after its translocation across the outer membrane using the recently developed SpyTag/SpyCatcher protein ligation system. As expected, fusion of the small SpyTag to Hbp did not hamper display on OMVs. Subsequent addition of purified proteins fused to the SpyCatcher domain resulted in efficient covalent coupling to Hbp-SpyTag. Using in addition the orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could be coupled to Hbp in a sequential ligation strategy. Not only antigens proved suitable for Spy-mediated ligation but also nanobodies. Addition of this functionality to the platform might allow the targeting of live bacterial or OMV vaccines to certain tissues or immune cells to tailor immune responses.IMPORTANCE Outer membrane vesicles (OMVs) derived from Gram-negative bacteria attract increasing interest in the development of vaccines and therapeutic agents. We aim to construct a semisynthetic OMV platform for recombinant antigen presentation on OMVs derived from attenuated Salmonella enterica serovar Typhimurium cells displaying an adapted Escherichia coli autotransporter, Hbp, at the surface. Although this autotransporter accepts substantial modifications, its capacity with respect to the number, size, and structural complexity of the antigens genetically fused to the Hbp carrier is restricted. Here we describe the application of SpyCatcher/SpyTag protein ligation technology to enzymatically link antigens to Hbp present at high density in OMVs. Protein ligation was apparently unobstructed by the membrane environment and allowed a high surface density of coupled antigens, a property we have shown to be important for vaccine efficacy. The OMV coupling procedure appears versatile and robust, allowing fast production of experimental vaccines and therapeutic agents through a modular plug-and-display procedure.

scholarly journals Attenuation of Salmonella enterica Serovar Typhimurium by Altering Biological Functions of Murein Lipoprotein and Lipopolysaccharide

2005 ◽  
Vol 73 (12) ◽  
pp. 8433-8436 ◽  
Author(s):  
A. A. Fadl ◽  
J. Sha ◽  
G. R. Klimpel ◽  
J. P. Olano ◽  
C. L. Galindo ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Biological Functions ◽  
Double Knockout ◽  
In Vivo Models ◽  
Background Strain ◽  
Knockout Mutants ◽  
Serovar Typhimurium

ABSTRACT We constructed Salmonella enterica serovar Typhimurium double-knockout mutants in which either the lipoprotein A (lppA) or the lipoprotein B (lppB) gene was deleted from an msbB-negative background strain by marker exchange mutagenesis. These mutants were highly attenuated when tested with in vitro and in vivo models of Salmonella pathogenesis.

Sign in / Sign up

Export Citation Format

Share Document