Structural Properties of Periplasmic SodCI That Correlate with Virulence in Salmonella enterica Serovar Typhimurium

ABSTRACT Salmonella enterica strains survive and propagate in macrophages by both circumventing and resisting the antibacterial effectors normally delivered to the phagosome. An important aspect of Salmonella resistance is the production of periplasmic superoxide dismutase to combat phagocytic superoxide. S. enterica serovar Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Both enzymes are produced during infection, but only SodCI contributes to virulence in the animal. Although 60% identical to SodCII at the amino acid level with very similar enzymatic properties, SodCI is dimeric, protease resistant, and tethered within the periplasm via a noncovalent interaction. In contrast, SodCII is monomeric and protease sensitive and is released from the periplasm normally by osmotic shock. We have constructed an enzymatically active monomeric SodCI enzyme by site-directed mutagenesis. The resulting protein was released by osmotic shock and sensitive to protease and could not complement the loss of wild-type dimeric SodCI during infection. To distinguish which property is most critical during infection, we cloned and characterized related SodC proteins from a variety of bacteria. Brucella abortus SodC was monomeric and released by osmotic shock but was protease resistant and could complement SodCI in the animal. These data suggest that protease resistance is a critical property that allows SodCI to function in the harsh environment of the phagosome to combat phagocytic superoxide. We propose a model to account for the various properties of SodCI and how they contribute to bacterial survival in the phagosome.

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Cytokine-Mediated Regulation of ARG1 in Macrophages and Its Impact on the Control of Salmonella enterica Serovar Typhimurium Infection

Cells ◽

10.3390/cells10071823 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1823

Author(s):

Natascha Brigo ◽

Christa Pfeifhofer-Obermair ◽

Piotr Tymoszuk ◽

Egon Demetz ◽

Sabine Engl ◽

...

Keyword(s):

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Natural Resistance ◽

Bacterial Survival ◽

Wild Type ◽

Salmonella Infections ◽

Arginase 1 ◽

Serovar Typhimurium ◽

The Impact

Arginase 1 (ARG1) is a cytosolic enzyme that cleaves L-arginine, the substrate of inducible nitric oxide synthase (iNOS), and thereby impairs the control of various intracellular pathogens. Herein, we investigated the role of ARG1 during infection with Salmonella enterica serovar Typhimurium (S.tm). To study the impact of ARG1 on Salmonella infections in vitro, bone marrow-derived macrophages (BMDM) from C57BL/6N wild-type, ARG1-deficient Tie2Cre+/−ARG1fl/fl and NRAMPG169 C57BL/6N mice were infected with S.tm. In wild-type BMDM, ARG1 was induced by S.tm and further upregulated by the addition of interleukin (IL)-4, whereas interferon-γ had an inhibitory effect. Deletion of ARG1 did not result in a reduction in bacterial numbers. In vivo, Arg1 mRNA was upregulated in the spleen, but not in the liver of C57BL/6N mice following intraperitoneal S.tm infection. The genetic deletion of ARG1 (Tie2Cre+/−ARG1fl/fl) or its pharmacological inhibition with CB-1158 neither affected the numbers of S.tm in spleen, liver and blood nor the expression of host response genes such as iNOS, IL-6 or tumour necrosis factor (TNF). Furthermore, ARG1 was dispensable for pathogen control irrespective of the presence or absence of the phagolysosomal natural resistance-associated macrophage protein 1 (NRAMP1). Thus, unlike the detrimental function of ARG1 seen during infections with other intraphagosomal microorganisms, ARG1 did not support bacterial survival in systemic salmonellosis, indicating differential roles of arginine metabolism for host immune response and microbe persistence depending on the type of pathogen.

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A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

The Journal of Cell Biology ◽

10.1083/jcb.200611056 ◽

2007 ◽

Vol 176 (3) ◽

pp. 263-268 ◽

Cited By ~ 107

Author(s):

Adam C. Smith ◽

Won Do Heo ◽

Virginie Braun ◽

Xiuju Jiang ◽

Chloe Macrae ◽

...

Keyword(s):

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Dominant Negative ◽

Rab Gtpases ◽

Wild Type ◽

Intracellular Growth ◽

Non Invasive ◽

Serovar Typhimurium ◽

Guanosine Triphosphatase ◽

Kinetics Of

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome–lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

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Salmonella enterica Serovar Typhimurium Uses PbgA/YejM To Regulate Lipopolysaccharide Assembly during Bacteremia

Infection and Immunity ◽

10.1128/iai.00758-19 ◽

2019 ◽

Vol 88 (1) ◽

Cited By ~ 16

Author(s):

Melina B. Cian ◽

Nicole P. Giordano ◽

Revathi Masilamani ◽

Keaton E. Minor ◽

Zachary D. Dalebroux

Keyword(s):

Salmonella Enterica ◽

Lipid A ◽

Salmonella Enterica Serovar Typhimurium ◽

Transmembrane Domain ◽

Wild Type ◽

Content Type ◽

Serovar Typhimurium ◽

Periplasmic Domain ◽

Inner Membrane Protein ◽

Substitution Mutations

ABSTRACT Salmonella enterica serovar Typhimurium (S. Typhimurium) relies upon the inner membrane protein PbgA to enhance outer membrane (OM) integrity and promote virulence in mice. The PbgA transmembrane domain (residues 1 to 190) is essential for viability, while the periplasmic domain (residues 191 to 586) is dispensable. Residues within the basic region (residues 191 to 245) bind acidic phosphates on polar phospholipids, like for cardiolipins, and are necessary for salmonella OM integrity. S. Typhimurium bacteria increase their OM cardiolipin concentrations during activation of the PhoPQ regulators. The mechanism involves PbgA’s periplasmic globular region (residues 245 to 586), but the biological role of increasing cardiolipins on the surface is not understood. Nonsynonymous polymorphisms in three essential lipopolysaccharide (LPS) synthesis regulators, lapB (also known as yciM), ftsH, and lpxC, variably suppressed the defects in OM integrity, rifampin resistance, survival in macrophages, and systemic colonization of mice in the pbgAΔ191–586 mutant (in which the PbgA periplasmic domain from residues 191 to 586 is deleted). Compared to the OMs of the wild-type salmonellae, the OMs of the pbgA mutants had increased levels of lipid A-core molecules, cardiolipins, and phosphatidylethanolamines and decreased levels of specific phospholipids with cyclopropanated fatty acids. Complementation and substitution mutations in LapB and LpxC generally restored the phospholipid and LPS assembly defects for the pbgA mutants. During bacteremia, mice infected with the pbgA mutants survived and cleared the bacteria, while animals infected with wild-type salmonellae succumbed within 1 week. Remarkably, wild-type mice survived asymptomatically with pbgA-lpxC salmonellae in their livers and spleens for months, but Toll-like receptor 4-deficient animals succumbed to these infections within roughly 1 week. In summary, S. Typhimurium uses PbgA to influence LPS assembly during stress in order to survive, adapt, and proliferate within the host environment.

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Differences in Enzymatic Properties Allow SodCI but Not SodCII To Contribute to Virulence in Salmonella enterica Serovar Typhimurium Strain 14028

Journal of Bacteriology ◽

10.1128/jb.186.16.5230-5238.2004 ◽

2004 ◽

Vol 186 (16) ◽

pp. 5230-5238 ◽

Cited By ~ 40

Author(s):

Radha Krishnakumar ◽

Maureen Craig ◽

James A. Imlay ◽

James M. Slauch

Keyword(s):

Salmonella Enterica ◽

Osmotic Shock ◽

Acid Resistance ◽

Noncovalent Interaction ◽

Open Reading Frames ◽

Striking Difference ◽

Significant Difference ◽

Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium produces two Cu/Zn cofactored periplasmic superoxide dismutases, SodCI and SodCII. While mutations in sodCI attenuate virulence eightfold, loss of SodCII does not confer a virulence phenotype, nor does it enhance the defect observed in a sodCI background. Despite this in vivo phenotype, SodCI and SodCII are expressed at similar levels in vitro during the stationary phase of growth. By exchanging the open reading frames of sodCI and sodCII, we found that SodCI contributes to virulence when placed under the control of the sodCII promoter. In contrast, SodCII does not contribute to virulence even when expressed from the sodCI promoter. Thus, the disparity in virulence phenotypes is due primarily to some physical difference between the two enzymes. In an attempt to identify the unique property of SodCI, we have tested factors that might affect enzyme activity inside a phagosome. We found no significant difference between SodCI and SodCII in their resistance to acid, resistance to hydrogen peroxide, or ability to obtain copper in a copper-limiting environment. Both enzymes are synthesized as apoenzymes in the absence of copper and can be fully remetallated when copper is added. The one striking difference that we noted is that, whereas SodCII is released normally by an osmotic shock, SodCI is “tethered” within the periplasm by an apparently noncovalent interaction. We propose that this novel property of SodCI is crucial to its ability to contribute to virulence in serovar Typhimurium.

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A Plasmid-Encoded FetMP-Fls Iron Uptake System Confers Selective Advantages to Salmonella enterica Serovar Typhimurium in Growth under Iron-Restricted Conditions and for Infection of Mammalian Host Cells

Microorganisms ◽

10.3390/microorganisms8050630 ◽

2020 ◽

Vol 8 (5) ◽

pp. 630

Author(s):

Vanesa García ◽

Ana Herrero-Fresno ◽

Rosaura Rodicio ◽

Alfonso Felipe-López ◽

Ignacio Montero ◽

...

Keyword(s):

Iron Content ◽

Salmonella Enterica ◽

Iron Uptake ◽

Type Strain ◽

Wild Type Strain ◽

Salmonella Enterica Serovar Typhimurium ◽

Iron Acquisition ◽

Clinical Strain ◽

Wild Type ◽

Serovar Typhimurium

The resistance plasmid pUO-StVR2, derived from virulence plasmid pSLT, is widespread in clinical isolates of Salmonella enterica serovar Typhimurium recovered in Spain and other European countries. pUO-StVR2 carries several genes encoding a FetMP-Fls system, which could be involved in iron uptake. We therefore analyzed S. Typhimurium LSP 146/02, a clinical strain selected as representative of the isolates carrying the plasmid, and an otherwise isogenic mutant lacking four genes (fetMP-flsDA) of the fetMP-fls region. Growth curves and determination of the intracellular iron content under iron-restricted conditions demonstrated that deletion of these genes impairs iron acquisition. Thus, under these conditions, the mutant grew significantly worse than the wild-type strain, its iron content was significantly lower, and it was outcompeted by the wild-type strain in competition assays. Importantly, the strain lacking the fetMP-flsDA genes was less invasive in cultured epithelial HeLa cells and replicated poorly upon infection of RAW264.7 macrophages. The genes were introduced into S. Typhimurium ATCC 14028, which lacks the FetMP-Fls system, and this resulted in increased growth under iron limitation as well as an increased ability to multiply inside macrophages. These findings indicate that the FetMP-Fls iron acquisition system exceeds the benefits conferred by the other high-affinity iron uptake systems carried by ATCC 14028 and LSP 146/02. We proposed that effective iron acquisition by this system in conjunction with antimicrobial resistance encoded from the same plasmid have greatly contributed to the epidemic success of S. Typhimurium isolates harboring pUO-StVR2.

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Substrate Recognition Properties of Oligopeptidase B from Salmonella enterica Serovar Typhimurium

Journal of Bacteriology ◽

10.1128/jb.184.12.3329-3337.2002 ◽

2002 ◽

Vol 184 (12) ◽

pp. 3329-3337 ◽

Cited By ~ 30

Author(s):

Rory E. Morty ◽

Vilmos Fülöp ◽

Norma W. Andrews

Keyword(s):

Substrate Specificity ◽

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Site Directed Mutagenesis ◽

Cloning And Expression ◽

Preferential Cleavage ◽

Oligopeptidase B ◽

Serovar Typhimurium ◽

Important Virulence Factor ◽

Catalytic Power

ABSTRACT Oligopeptidase B (OpdB) is a serine peptidase broadly distributed among unicellular eukaryotes, gram-negative bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases. We report here the cloning and expression of the opdB homologue from Salmonella enterica serovar Typhimurium and demonstrate that it exhibits amidolytic activity exclusively against substrates with basic residues in P1. While similar to its eukaryotic homologues in terms of substrate specificity, Salmonella OpdB differs significantly in catalytic power and inhibition and activation properties. In addition to oligopeptide substrates, restricted proteolysis of histone proteins was observed, although no cleavage was seen at or near residues that had been posttranslationally modified or at defined secondary structures. This supports the idea that the catalytic site of OpdB may be accessible only to unstructured oligopeptides, similar to the closely related prolyl oligopeptidase (POP). Salmonella OpdB was employed as a model enzyme to define determinants of substrate specificity that distinguish OpdB from POP, which hydrolyzes substrates exclusively at proline residues. Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs but absent from POPs were converted to their corresponding residues in POP. In this manner, we identified a pair of glutamic acid residues, Glu576 and Glu578, that define P1 specificity and direct OpdB cleavage C terminal to basic residues. We have also identified a second pair of residues, Asp460 and Asp462, that may be involved in defining P2 specificity and thus direct preferential cleavage by OpdB after pairs of basic residues.

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Role of the alternative sigma factors σ E and σ S in survival of Salmonella enterica serovar Typhimurium during starvation, refrigeration and osmotic shock

Microbiology ◽

10.1099/mic.0.29235-0 ◽

2007 ◽

Vol 153 (1) ◽

pp. 263-269 ◽

Cited By ~ 39

Author(s):

Alisdair McMeechan ◽

Mark Roberts ◽

Tristan A. Cogan ◽

Frieda Jørgensen ◽

Andrew Stevenson ◽

...

Keyword(s):

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Osmotic Shock ◽

Sigma Factors ◽

Alternative Sigma Factors ◽

Serovar Typhimurium

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sciS, an icmF Homolog in Salmonella enterica Serovar Typhimurium, Limits Intracellular Replication and Decreases Virulence

Infection and Immunity ◽

10.1128/iai.73.7.4338-4345.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4338-4345 ◽

Cited By ~ 93

Author(s):

Duncan A. Parsons ◽

Fred Heffron

Keyword(s):

Legionella Pneumophila ◽

Salmonella Enterica ◽

Wild Type Strain ◽

Salmonella Enterica Serovar Typhimurium ◽

The Body ◽

Wild Type ◽

Serovar Typhimurium ◽

Intracellular Multiplication ◽

Late Stages ◽

Made In

ABSTRACT Salmonella enterica serovar Typhimurium utilizes macrophages to disseminate from the intestine to deeper tissues within the body. While S. enterica serovar Typhimurium has been shown to kill its host macrophage, it can persist intracellularly beyond 18 h postinfection. To identify factors involved in late stages of infection, we screened a transposon library made in S. enterica serovar Typhimurium for the ability to persist in J774 macrophages at 24 h postinfection. Through this screen, we identified a gene, sciS, found to be homologous to icmF in Legionella pneumophila. icmF, which is required for intracellular multiplication, is conserved in several gram-negative pathogens, and its homolog appears to have been acquired horizontally in S. enterica serovar Typhimurium. We found that an sciS mutant displayed increased intracellular numbers in J774 macrophages when compared to the wild-type strain at 24 h postinfection. sciS was maximally transcribed at 27 h postinfection and is repressed by SsrB, an activator of genes required for promoting intracellular survival. Finally, we demonstrate that an sciS mutant is hypervirulent in mice when administered intragastrically. Taken together, these data indicate a role for SciS in controlling intracellular bacterial levels at later stages of infection and attenuating virulence in a murine host

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Effect of Inactivation of degS on Salmonella enterica Serovar Typhimurium In Vitro and In Vivo

Infection and Immunity ◽

10.1128/iai.73.1.459-463.2005 ◽

2005 ◽

Vol 73 (1) ◽

pp. 459-463 ◽

Cited By ~ 23

Author(s):

Gary Rowley ◽

Andrew Stevenson ◽

Jan Kormanec ◽

Mark Roberts

Keyword(s):

Sigma Factor ◽

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Wild Type ◽

Alternative Pathways ◽

E Coli ◽

Mammalian Tissues ◽

Serovar Typhimurium

ABSTRACT The alternative sigma factor (RpoE σE) enables Salmonella enterica serovar Typhimurium to adapt to stressful conditions, such as oxidative stress, nutrient deprivation, and growth in mammalian tissues. Infection of mice by Salmonella serovar Typhimurium also requires σE. In Escherichia coli, activation of the σE pathway is dependent on proteolysis of the anti-sigma factor RseA and is initiated by DegS. DegS is also important in order for E. coli to cause extraintestinal infection in mice. We constructed a degS mutant of the serovar Typhimurium strain SL1344 and compared its behavior in vitro and in vivo with those of its wild-type (WT) parent and an isogenic rpoE mutant. Unlike E. coli degS strains, the Salmonella serovar Typhimurium degS strain grew as well as the WT strain at 42°C. The degS mutant survived very poorly in murine macrophages in vitro and was highly attenuated compared with the WT strain for both the oral and parenteral routes of infection in mice. However, the degS mutant was not as attenuated as the serovar Typhimurium rpoE mutant: 100- to 1,000-fold more degS bacteria than rpoE bacteria were present in the livers and spleens of mice 24 h after intraperitoneal challenge. In most assays, the rpoE mutant was more severely affected than the degS mutant and a σE-dependent reporter gene was more active in the degS mutant than the rpoE strain. These findings indicate that degS is important for activation of the σE pathway in serovar Typhimurium but that alternative pathways for σE activation probably exist.

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