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

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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Palmitoylation State Impacts Induction of Innate and Acquired Immunity by the Salmonella enterica Serovar TyphimuriummsbBMutant

Infection and Immunity ◽

10.1128/iai.05524-11 ◽

2011 ◽

Vol 79 (12) ◽

pp. 5027-5038 ◽

Cited By ~ 25

Author(s):

Qingke Kong ◽

David A. Six ◽

Qing Liu ◽

Lillian Gu ◽

Kenneth L. Roland ◽

...

Keyword(s):

Salmonella Enterica ◽

Lipid A ◽

Inflammatory Responses ◽

Outer Membrane Proteins ◽

Acyl Chain ◽

Wild Type ◽

Proinflammatory Response ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTLipopolysaccharide (LPS), composed of lipid A, core, and O-antigen, is a major virulence factor ofSalmonella entericaserovar Typhimurium, with lipid A being a major stimulator to induce the proinflammatory response via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. WhileSalmonella msbBmutants lacking the myristate chain in lipid A were investigated widely as an anticancer vaccine, inclusion of themsbBmutation in aSalmonellavaccine to deliver heterologous antigens has not yet been investigated. We introduced themsbBmutation alone or in combination with mutations in other lipid A acyl chain modification genes encoding PagL, PagP, and LpxR into wild-typeS. entericaserovar Typhimurium. ThemsbBmutation reduced virulence, while thepagL,pagP, andlpxRmutations did not affect virulence in themsbBmutant background when administered orally to BALB/c mice. Also, all mutants exhibited sensitivity to polymyxin B but did not display sensitivity to deoxycholate. LPS derived frommsbBmutants induced less inflammatory responses in human Mono Mac 6 and murine macrophage RAW264.7 cellsin vitro. However, anmsbBmutant did not decrease the induction of inflammatory responses in mice compared to the levels induced by the wild-type strain, whereas anmsbB pagPmutant induced less inflammatory responsesin vivo. The mutations were moved to an attenuatedSalmonellavaccine strain to evaluate their effects on immunogenicity. Lipid A modification caused by themsbBmutation alone and in combination withpagL,pagP, andlpxRmutations led to higher IgA production in the vaginal tract but still retained the same IgG titer level in serum to PspA, a test antigen fromStreptococcus pneumoniae, and to outer membrane proteins (OMPs) fromSalmonella.

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Effect of Deletion of Genes Involved in Lipopolysaccharide Core and O-Antigen Synthesis on Virulence and Immunogenicity of Salmonella enterica Serovar Typhimurium

Infection and Immunity ◽

10.1128/iai.05398-11 ◽

2011 ◽

Vol 79 (10) ◽

pp. 4227-4239 ◽

Cited By ~ 106

Author(s):

Qingke Kong ◽

Jiseon Yang ◽

Qing Liu ◽

Praveen Alamuri ◽

Kenneth L. Roland ◽

...

Keyword(s):

Salmonella Enterica ◽

Lipid A ◽

Outer Core ◽

Wild Type ◽

Core Oligosaccharide ◽

Content Type ◽

O Antigen ◽

Serovar Typhimurium

ABSTRACTLipopolysaccharide (LPS) is a major virulence factor ofSalmonella entericaserovar Typhimurium and is composed of lipid A, core oligosaccharide (C-OS), and O-antigen polysaccharide (O-PS). While the functions of the gene products involved in synthesis of core and O-antigen have been elucidated, the effect of removing O-antigen and core sugars on the virulence and immunogenicity ofSalmonella entericaserovar Typhimurium has not been systematically studied. We introduced nonpolar, defined deletion mutations inwaaG(rfaG),waaI(rfaI),rfaH,waaJ(rfaJ),wbaP(rfbP),waaL(rfaL), orwzy(rfc) into wild-typeS.Typhimurium. The LPS structure was confirmed, and a number ofin vitroandin vivoproperties of each mutant were analyzed. All mutants were significantly attenuated compared to the wild-type parent when administered orally to BALB/c mice and were less invasive in host tissues. Strains with ΔwaaGand ΔwaaImutations, in particular, were deficient in colonization of Peyer's patches and liver. This deficiency could be partially overcome in the ΔwaaImutant when it was administered intranasally. In the context of an attenuated vaccine strain delivering the pneumococcal antigen PspA, all of the mutations tested resulted in reduced immune responses against PspA andSalmonellaantigens. Our results indicate that nonreversible truncation of the outer core is not a viable option for developing a live oralSalmonellavaccine, while awzymutant that retains one O-antigen unit is adequate for stimulating the optimal protective immunity to homologous or heterologous antigens by oral, intranasal, or intraperitoneal routes of administration.

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Extracellular Loops of Lipid A 3-O-Deacylase PagL Are Involved in Recognition of Aminoarabinose-Based Membrane Modifications in Salmonella enterica Serovar Typhimurium

Journal of Bacteriology ◽

10.1128/jb.00587-08 ◽

2008 ◽

Vol 190 (16) ◽

pp. 5597-5606 ◽

Cited By ~ 15

Author(s):

Takayuki Manabe ◽

Kiyoshi Kawasaki

Keyword(s):

Amino Acid ◽

Salmonella Enterica ◽

Lipid A ◽

Salmonella Enterica Serovar Typhimurium ◽

Point Mutations ◽

Amino Acid Residues ◽

Wild Type ◽

Extracellular Loops ◽

Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium modifies its lipopolysaccharide (LPS), including the lipid A portion, in response to changes in its environment including host tissues. The lipid A 3-O-deacylase PagL, the expression of which is promoted under a host-mimetic environment, exhibits latency in S. enterica; deacylation of lipid A is not usually observed in vivo, despite the expression of the outer membrane protein PagL. In contrast, PagL does not exhibit latency in S. enterica pmrA and pmrE mutants, both of which are deficient in the aminoarabinose-based modification of lipid A, indicating that aminoarabinose-modified LPS species were involved in the latency. In order to analyze the machinery for PagL's repression, we generated PagL mutants in which an amino acid residue located at four extracellular loops was replaced with alanine. Apparent lipid A 3-O deacylation was observed in S. enterica expressing the recombinant mutants PagL(R43A), PagL(R44A), PagL(C85A), and PagL(R135A), but not in S. enterica expressing wild-type PagL, suggesting that the point mutations released PagL from the latency. In addition, mutations at Arg-43, Arg-44, Cys-85, and Arg-135 did not affect lipid A 3-O-deacylase activity in an S. enterica pmrA mutant or in Escherichia coli BL21(DE3). These results, taken together, indicate that specific amino acid residues located at extracellular loops of PagL are involved in the recognition of aminoarabinose-modified LPS. Furthermore, S. enterica expressing the recombinant PagL(R43A) or PagL(R135A) mutant showed apparent growth arrest at 43°C compared with S. enterica expressing wild-type PagL, indicating that the latency of PagL is important for bacterial growth.

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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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Regulation of RamA by RamR in Salmonella enterica Serovar Typhimurium: Isolation of a RamR Superrepressor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01320-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 6037-6040 ◽

Cited By ~ 15

Author(s):

Vito Ricci ◽

Stephen J. W. Busby ◽

Laura J. V. Piddock

Keyword(s):

Transcription Factor ◽

Salmonella Enterica ◽

Fluorescent Protein ◽

Salmonella Enterica Serovar Typhimurium ◽

Promoter Sequence ◽

Palindromic Sequence ◽

Content Type ◽

Gfp Reporter ◽

Serovar Typhimurium ◽

Green Fluorescent

ABSTRACTRamA is a transcription factor involved in regulating multidrug resistance inSalmonella entericaserovar Typhimurium SL1344. Green fluorescent protein (GFP) reporter fusions were exploited to investigate the regulation of RamA expression by RamR. We show that RamR represses theramApromoter by binding to a palindromic sequence and describe a superrepressor RamR mutant that binds to theramApromoter sequence more efficiently, thus exhibiting aramAinactivated phenotype.

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Global Transcriptional Analysis of Dehydrated Salmonella enterica Serovar Typhimurium

Applied and Environmental Microbiology ◽

10.1128/aem.01822-12 ◽

2012 ◽

Vol 78 (22) ◽

pp. 7866-7875 ◽

Cited By ~ 48

Author(s):

Nadia Gruzdev ◽

Michael McClelland ◽

Steffen Porwollik ◽

Shany Ofaim ◽

Riky Pinto ◽

...

Keyword(s):

Salmonella Enterica ◽

Lipid A ◽

Isocitrate Lyase ◽

Protein Biosynthesis ◽

Potassium Transport ◽

Transcriptional Analysis ◽

Scaffolding Protein ◽

Transport Channel ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTDespite the scientific and industrial importance of desiccation tolerance inSalmonella, knowledge regarding its genetic basis is still scarce. In the present study, we performed a transcriptomic analysis of dehydrated and water-suspendedSalmonella entericaserovar Typhimurium using microarrays. Dehydration induced expression of 90 genes and downregulated that of 7 genes. Ribosomal structural genes represented the most abundant functional group with a relatively higher transcription during dehydration. Other main induced functional groups included genes involved in amino acid metabolism, energy production, ion transport, transcription, and stress response. The highest induction was observed in thekdpFABCoperon, encoding a potassium transport channel. Knockout mutations were generated in nine upregulated genes. Five mutants displayed lower tolerance to desiccation, implying the involvement of the corresponding genes in the adaptation ofSalmonellato desiccation. These included genes encoding the isocitrate-lyase AceA, the lipid A biosynthesis palmitoleoyl-acyltransferase Ddg, the modular iron-sulfur cluster scaffolding protein NifU, the global regulator Fnr, and the alternative sigma factor RpoE. Notably, these proteins were previously implicated in the response ofSalmonellato oxidative stress, heat shock, and cold shock. A strain with a mutation in the structural genekdpAhad a tolerance to dehydration comparable to that of the parent strain, implying that potassium transport through this system is dispensable for early adaptation to the dry environment. Nevertheless, this mutant was significantly impaired in long-term persistence during cold storage. Our findings indicate the involvement of a relatively small fraction of theSalmonellagenome in transcriptional adjustment from water to dehydration, with a high prevalence of genes belonging to the protein biosynthesis machinery.

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SlyA and HilD Counteract H-NS-Mediated Repression on the ssrAB Virulence Operon of Salmonella enterica Serovar Typhimurium and Thus Promote Its Activation by OmpR

Journal of Bacteriology ◽

10.1128/jb.00530-18 ◽

2019 ◽

Vol 201 (8) ◽

Cited By ~ 3

Author(s):

María M. Banda ◽

Crispín Zavala-Alvarado ◽

Deyanira Pérez-Morales ◽

Víctor H. Bustamante

Keyword(s):

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Growth Conditions ◽

The Other ◽

Regulatory Mechanisms ◽

Two Component System ◽

Content Type ◽

Additive Action ◽

Serovar Typhimurium ◽

Bacterial Fitness

ABSTRACT H-NS-mediated repression of acquired genes and the subsequent adaptation of regulatory mechanisms that counteract this repression have played a central role in the Salmonella pathogenicity evolution. The Salmonella pathogenicity island 2 (SPI-2) is an acquired chromosomal region containing genes necessary for Salmonella enterica to colonize and replicate in different niches of hosts. The ssrAB operon, located in SPI-2, encodes the two-component system SsrA-SsrB, which positively controls the expression of the SPI-2 genes but also other many genes located outside SPI-2. Several regulators have been involved in the expression of ssrAB, such as the ancestral regulators SlyA and OmpR, and the acquired regulator HilD. In this study, we show how SlyA, HilD, and OmpR coordinate to induce the expression of ssrAB under different growth conditions. We found that when Salmonella enterica serovar Typhimurium is grown in nutrient-rich lysogeny broth (LB), SlyA and HilD additively counteract H-NS-mediated repression on ssrAB, whereas in N-minimal medium (N-MM), SlyA antagonizes H-NS-mediated repression on ssrAB independently of HilD. Interestingly, our results indicate that OmpR is required for the expression of ssrAB independently of the growth conditions, even in the absence of repression by H-NS. Therefore, our data support two mechanisms adapted for the expression of ssrAB under different growth conditions. One involves the additive action of SlyA and HilD, whereas the other involves SlyA, but not HilD, to counteract H-NS-mediated repression on ssrAB, thus favoring in both cases the activation of ssrAB by OmpR. IMPORTANCE The global regulator H-NS represses the expression of acquired genes and thus avoids possible detrimental effects on bacterial fitness. Regulatory mechanisms are adapted to induce expression of the acquired genes in particular niches to obtain a benefit from the information encoded in the foreign DNA, as for pathogenesis. Here, we show two mechanisms that were integrated for the expression of virulence genes in Salmonella Typhimurium. One involves the additive action of the regulators SlyA and HilD, whereas the other involves SlyA, but not HilD, to counteract H-NS-mediated repression on the ssrAB operon, thus favoring its activation by the OmpR regulator. To our knowledge, this is the first report involving the coordinated action of two regulators to counteract H-NS-mediated repression.

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Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Siphophage Siskin

Microbiology Resource Announcements ◽

10.1128/mra.00188-19 ◽

2019 ◽

Vol 8 (18) ◽

Author(s):

Chandler O’Leary ◽

Yicheng Xie ◽

Rohit Kongari ◽

Jason J. Gill ◽

Mei Liu

Keyword(s):

Genome Sequence ◽

Complete Genome Sequence ◽

Salmonella Enterica ◽

Complete Genome ◽

Salmonella Enterica Serovar Typhimurium ◽

Content Type ◽

New Class ◽

Typhimurium Strain ◽

Serovar Typhimurium ◽

Lysis Cassette

Bacteriophage Siskin is a member of the χ-like siphovirus phage cluster that infects Salmonella enterica serovar Typhimurium strain LT2. Here, we report the complete 58,476-bp sequence of the Siskin genome, provide confirmation of its genomic termini, and describe a potentially new class of holins and endolysins found in the lysis cassette.

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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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Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Siphophage Skate

Microbiology Resource Announcements ◽

10.1128/mra.00541-19 ◽

2019 ◽

Vol 8 (27) ◽

Author(s):

Matthew Rohren ◽

Yicheng Xie ◽

Chandler O’Leary ◽

Rohit Kongari ◽

Jason Gill ◽

...

Keyword(s):

Genome Sequence ◽

Complete Genome Sequence ◽

Salmonella Enterica ◽

Complete Genome ◽

Salmonella Enterica Serovar Typhimurium ◽

Foodborne Illnesses ◽

Gram Negative ◽

Content Type ◽

Typhimurium Strain ◽

Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium is a Gram-negative pathogen and a primary cause of foodborne illnesses worldwide. Here, we present the complete 47,393-bp genome sequence of the siphophage Skate, which was isolated against S. Typhimurium strain LT2.

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