scholarly journals Inhibition of Salmonella enterica Serovar Typhimurium Lipopolysaccharide Deacylation by Aminoarabinose Membrane Modification

2005 ◽  
Vol 187 (7) ◽  
pp. 2448-2457 ◽  
Author(s):  
Kiyoshi Kawasaki ◽  
Robert K. Ernst ◽  
Samuel I. Miller
Keyword(s):  
Outer Membrane ◽  
Salmonella Enterica ◽  
Lipid A ◽  
Bacterial Resistance ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Host Recognition ◽  
Membrane Modification ◽  
Serovar Typhimurium ◽  
Nonionic Detergent

ABSTRACT Salmonella enterica serovar Typhimurium remodels the lipid A component of lipopolysaccharide, a major component of the outer membrane, to survive within animals. The activation of the sensor kinase PhoQ in host environments increases the synthesis of enzymes that deacylate, palmitoylate, hydroxylate, and attach aminoarabinose to lipid A, also known as endotoxin. These modifications promote bacterial resistance to antimicrobial peptides and reduce the host recognition of lipid A by Toll-like receptor 4. The Salmonella lipid A 3-O-deacylase, PagL, is an outer membrane protein whose expression is regulated by PhoQ. In S. enterica serovar Typhimurium strains that had the ability to add aminoarabinose to lipid A, 3-O-deacylated lipid A species were not detected, despite the PhoQ induction of PagL protein expression. In contrast, strains defective for the aminoarabinose modification of lipid A demonstrated in vivo PagL activity, indicating that this membrane modification inhibited PagL's enzymatic activity. Since not all lipid A molecules are modified with aminoarabinose upon PhoQ activation, these results cannot be ascribed to the substrate specificity of PagL. PagL-dependent deacylation was detected in sonically disrupted membranes and membranes treated with the nonionic detergent n-octyl-β-d-glucopyranoside, suggesting that perturbation of the intact outer membrane releases PagL from posttranslational inhibition by aminoarabinose-containing membranes. Taken together, these results suggest that PagL enzymatic deacylation is posttranslationally inhibited by membrane environments, which either sequester PagL from its substrate or alter its conformation.

scholarly journals Identification of cptA, a PmrA-Regulated Locus Required for Phosphoethanolamine Modification of the Salmonella enterica Serovar Typhimurium Lipopolysaccharide Core

2005 ◽  
Vol 187 (10) ◽  
pp. 3391-3399 ◽  
Author(s):  
R. Tamayo ◽  
B. Choudhury ◽  
A. Septer ◽  
M. Merighi ◽  
R. Carlson ◽  
...  
Keyword(s):  
Surface Charge ◽  
Salmonella Enterica ◽  
Lipid A ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Regulatory System ◽  
Polymyxin B ◽  
Serovar Typhimurium ◽  
Antimicrobial Peptide Resistance

ABSTRACT In response to the in vivo environment, the Salmonella enterica serovar Typhimurium lipopolysaccharide (LPS) is modified. These modifications are controlled in part by the two-component regulatory system PmrA-PmrB, with the addition of 4-aminoarabinose (Ara4N) to the lipid A and phosphoethanolamine (pEtN) to the lipid A and core. Here we demonstrate that the PmrA-regulated STM4118 (cptA) gene is necessary for the addition of pEtN to the LPS core. pmrC, a PmrA-regulated gene necessary for the addition of pEtN to lipid A, did not affect core pEtN addition. Although imparting a similar surface charge modification as Ara4N, which greatly affects polymyxin B resistance and murine virulence, neither pmrC nor cptA plays a dramatic role in antimicrobial peptide resistance in vitro or virulence in the mouse model. Therefore, factors other than surface charge/electrostatic interaction contribute to resistance to antimicrobial peptides such as polymyxin B.

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.

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

2008 ◽  
Vol 190 (16) ◽  
pp. 5597-5606 ◽  
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.

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

2019 ◽  
Vol 88 (1) ◽  
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.

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.

scholarly journals Yersinia pestis Is Viable with Endotoxin Composed of Only Lipid A

2005 ◽  
Vol 187 (18) ◽  
pp. 6599-6600 ◽  
Author(s):  
Li Tan ◽  
Creg Darby
Keyword(s):  
Escherichia Coli ◽  
Yersinia Pestis ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Lipid A ◽  
Gram Negative Bacteria ◽  
Membrane Component ◽  
Gram Negative ◽  
Serovar Typhimurium

ABSTRACT Lipopolysaccharide (LPS) is the major outer membrane component of gram-negative bacteria. The minimal LPS structure for viability of Escherichia coli and Salmonella enterica serovar Typhimurium is lipid A glycosylated with 3-deoxy-D-manno-octulosonic acid (Kdo) residues. Here we show that another member of the Enterobacteriaceae, Yersinia pestis, can survive without Kdo in its LPS.

scholarly journals The enzyme phosphoglucomutase (Pgm) is required by Salmonella enterica serovar Typhimurium for O-antigen production, resistance to antimicrobial peptides and in vivo fitness

Microbiology ◽  
2009 ◽  
Vol 155 (10) ◽  
pp. 3403-3410 ◽  
Author(s):  
G. K. Paterson ◽  
D. B. Cone ◽  
S. E. Peters ◽  
D. J. Maskell
Keyword(s):  
Antimicrobial Peptides ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Live Attenuated Vaccine ◽  
Antigen Production ◽  
O Antigen ◽  
Serovar Typhimurium

The enzyme phosphoglucomutase (Pgm) catalyses the interconversion of glucose 1-phosphate and glucose 6-phosphate and contributes to glycolysis and the generation of sugar nucleotides for biosynthesis. To assess the role of this enzyme in the biology of the pathogen Salmonella enterica serovar Typhimurium we have characterized a pgm deletion mutant in strain SL1344. Compared to SL1344, SL1344 pgm had impaired growth in vitro, was deficient in the ability to utilize galactose as a carbon source and displayed reduced O-antigen polymer length. The mutant was also more susceptible to antimicrobial peptides and showed decreased fitness in the mouse typhoid model. The in vivo phenotype of SL1344 pgm indicated a role for pgm in the early stages of infection, most likely through deficient O-antigen production. Although pgm mutants in other pathogens have potential as live attenuated vaccine strains, SL1344 pgm was not sufficiently attenuated for such use.

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