Conserved tandem arginines for PbgA/YejM allow Salmonella Typhimurium to regulate LpxC and control lipopolysaccharide biogenesis during infection.

2021 ◽  
Author(s):  
Nicole P. Giordano ◽  
Joshua A. Mettlach ◽  
Zachary D. Dalebroux
Keyword(s):  
Lipid A ◽  
Systemic Disease ◽  
Alpha Helix ◽  
Transmembrane Segments ◽  
Ftsh Protease ◽  
Serovar Typhimurium ◽  
Inner Membrane Protein ◽  
Key Residues ◽  
And Control ◽  
Basic Region

Enterobacteriaceae use the periplasmic domain of the conserved inner membrane protein, PbgA/YejM, to regulate lipopolysaccharide (LPS) biogenesis. Salmonella enterica serovar Typhimurium ( S. Typhimurium) relies on PbgA to cause systemic disease in mice and this involves functional interactions with LapB/YciM, FtsH, and LpxC. Escherichia coli PbgA interacts with LapB, an adaptor for the FtsH protease, via the transmembrane segments. LapB and FtsH control proteolysis of LpxC, the rate-limiting LPS biosynthesis enzyme. Lipid A-core, the hydrophobic anchor of LPS molecules, co-crystallizes with PbgA and interacts with residues in the basic region. The model predicts that PbgA-LapB detects periplasmic LPS molecules and prompts FtsH to degrade LpxC. However, the key residues and critical interactions are not defined. We establish that S. Typhimurium uses PbgA to regulate LpxC and define the contribution of two pairs of arginines within the basic region. PbgA R215 R216 form contacts with lipid A-core in the structure and R231 R232 exist in an adjacent alpha helix. PbgA R215 R216 are necessary for S . Typhimurium to regulate LpxC, control lipid-A core biogenesis, promote survival in macrophages, and enhance virulence in mice. In contrast, PbgA R231 R232 are not necessary to regulate LpxC or to control lipid A-core levels, nor are they necessary to promote survival in macrophages or mice. However, residues R231 R232 are critical for infection lethality, and the persistent infection phenotype requires mouse Toll-like receptor four, which detects lipid A. Therefore, S. Typhimurium relies on PbgA’s tandem arginines for multiple interconnected mechanisms of LPS regulation that enhance pathogenesis.

scholarly journals Conserved tandem arginines for PbgA/YejM allow Salmonella to regulate LpxC and control lipopolysaccharide biogenesis during infection

2020 ◽  
Author(s):  
Nicole P. Giordano ◽  
Joshua A. Mettlach ◽  
Zachary D. Dalebroux
Keyword(s):  
Outer Membrane ◽  
Salmonella Enterica ◽  
Lipid A ◽  
Transmembrane Protein ◽  
Systemic Disease ◽  
Side Chain ◽  
Serovar Typhimurium ◽  
And Control ◽  
Rate Limiting ◽  
Severe Systemic Disease

ABSTRACTSalmonella enterica serovar Typhimurium uses PbgA/YejM, a conserved multi-pass transmembrane protein with a soluble periplasmic domain (PD), to balance the glycerophospholipid (GPL) and lipopolysaccharide (LPS) concentrations within the outer membrane (OM). The lipid homeostasis and virulence defects of pbgAΔ191-586 mutants, which are deleted for the PD, can be suppressed by substitutions in three LPS regulators, LapB/YciM, FtsH, and LpxC. We reasoned that S. Typhimurium uses the PbgA PD to regulate LpxC through functional interactions with LapB and FtsH. In the stationary phase of growth, pbgAΔ191-586 mutants accumulated LpxC and overproduced LPS precursors, known as lipid A-core molecules. Trans-complementation fully decreased the LpxC and lipid A-core levels for the mutants, while substitutions in LapB, FtsH, and LpxC variably reduced the concentrations. PbgA binds lipid A-core, in part, using dual arginines, R215 and R216, which are located near the plasma membrane. Neutral, conservative, and non-conservative substitutions were engineered at these positions to test whether the side-chain charges for residues 215 and 216 influenced LpxC regulation. Salmonellae that expressed PbgA with dual alanines or aspartic acids overproduced LpxC, accumulated lipid A-core and short-LPS molecules, and were severely attenuated in mice. Bacteria that expressed PbgA with tandem lysines were fully virulent in mice and yielded LpxC and lipid A-core levels that were similar to the wild type. Thus, S. Typhimurium uses the cationic charge of PbgA R215 and R216 to down-regulate LpxC and decrease lipid A-core biosynthesis in response to host stress and this regulatory mechanism enhances their virulence during bacteremia.IMPORTANCESalmonella enterica serovar Typhimurium causes self-limiting gastroenteritis in healthy individuals and severe systemic disease in immunocompromised humans. The pathogen manipulates the immune system of its host by regulating the lipid, protein, and polysaccharide content of the outer membrane (OM) bilayer. Lipopolysaccharides (LPS) comprise the external leaflet of the OM, and are essential for establishing the OM barrier and providing gram-negative microbes with intrinsic antimicrobial resistance. LPS molecules are potent endotoxins and immunomodulatory ligands that bind host-pattern receptors, which control host resistance and adaptation during infection. Salmonellae use the cationic charge of dual arginines for PbgA/YejM to negatively regulate LPS biosynthesis. The mechanism involves PbgA binding to an LPS precursor and activating a conserved multi-protein signal transduction network that cues LpxC proteolysis, the rate-limiting enzyme. The cationic charge of the tandem arginines is critical for the ability of salmonellae to survive intracellularly and to cause systemic disease in mice.

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 Effects of Camu-Camu (Myrciaria dubia) Powder on the Physicochemical and Kinetic Parameters of Deteriorating Microorganisms and Salmonella enterica Subsp. enterica Serovar Typhimurium in Refrigerated Vacuum-Packed Ground Beef

Agriculture ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 252
Author(s):  
Jorge Luiz da Silva ◽  
Vasco Cadavez ◽  
José M. Lorenzo ◽  
Eduardo Eustáquio de Souza Figueiredo ◽  
Ursula Gonzales-Barron
Keyword(s):  
Ground Beef ◽  
Treated Group ◽  
B Value ◽  
Kinetics Parameters ◽  
Psychrotrophic Bacteria ◽  
Color Changes ◽  
A Value ◽  
Spoilage Bacteria ◽  
Serovar Typhimurium ◽  
And Control

This study aims to evaluate the effects of camu-camu powder (CCP), Amazonian berry fruit with documented bioactive properties, physicochemical meat parameters, and the growth kinetics parameters of S. enterica ser. Typhimurium, psychrotrophic bacteria (PSY), and lactic acid bacteria (LAB) in vacuum-packed ground beef. Batches of ground beef were mixed with 0.0%, 2.0%, 3.5%, and 5.0% CCP (w/w), vacuum-packed as 10 g portions, and stored at 5 °C for 16 days. Centesimal composition analyses (only on the initial day), pH, TBARS, and color were quantified on storage days 1, 7, and 15, while PSY and LAB were counted on days 0, 3, 6, 9, 13, and 16. Another experiment was conducted with the same camu-camu doses by inoculating S. enterica ser. Typhimurium microbial kinetic curves were modeled by the Huang growth and Weibull decay models. CCP decreased TBARS in beef from 0.477 to 0.189 mg MDA·kg−1. No significant differences in meat pH between treated and control samples were observed on day 15. CCP addition caused color changes, with color a* value decreases (from 14.45 to 13.44) and color b* value increases (from 17.41 to 21.25), while color L* was not affected. Higher CCP doses caused progressive LAB growth inhibition from 0.596 to 0.349 log CFU·day−1 at 2.0% and 5.0% CCP, respectively. Similarly, PSY growth rates in the treated group were lower (0.79–0.91 log CFU·day−1) compared to the control (1.21 log CFU·day−1). CCP addition at any of the investigated doses produced a steeper S. enterica ser. Typhimurium inactivation during the first cold storage day, represented by Weibull’s concavity α shape parameter, ranged from 0.37 to 0.51, in contrast to 1.24 for the control. At the end of the experiment, however, S. enterica ser. Typhimurium counts in beef containing CCP were not significantly different (p < 0.05) from the control. Although CCP affects bacterial kinetics, it does not protect ground beef against spoilage bacteria and Salmonella to the same degree it does against lipid peroxidation.

scholarly journals Focus on localized laryngeal amyloidosis: management of five cases

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 327-332
Author(s):  
Massimo Mesolella ◽  
Gerardo Petruzzi ◽  
Sarah Buono ◽  
Grazia Salerno ◽  
Francesco Antonio Salzano ◽  
...  
Keyword(s):  
Systemic Disease ◽  
Clinical Aspects ◽  
Benign Tumors ◽  
Local Form ◽  
Systemic Involvement ◽  
Clinical Syndromes ◽  
Laryngeal Amyloidosis ◽  
And Control ◽  
Localized Laryngeal Amyloidosis

AbstractAmyloidosis is a group of idiopathic clinical syndromes caused by the deposition of insoluble fibrillar proteins (amyloid) in the extracellular matrix of organs and tissues. These deposits disrupt the function of the target organ. Amyloidosis can manifest as a systemic disease or a single-organ involvement (local form). Its etiology still remains unclear. Deposits of amyloid in the larynx are rare, accounting for between 0.2 and 1.2% of benign tumors of the larynx. In this retrospective study, we report the clinical aspects, diagnosis, treatment and follow-up of five female patients with localized laryngeal amyloidosis without systemic involvement. The patients were all treated successfully using microlaryngoscopy with CO2 laser or cold instruments. Prognosis is excellent; however, appropriate follow-up is an important part of the long-term management of this disease in order to prevent and control the possibility of local recurrence.

scholarly journals Potential Compounds for the Inhibition of TMPRSS2

2020 ◽  
Author(s):  
Muhammad Roomi ◽  
Yaser Khan
Keyword(s):  
In Silico Analysis ◽  
Chemical Compounds ◽  
Vital Role ◽  
Large Sets ◽  
Potential Inhibitors ◽  
Key Residues ◽  
And Control ◽  
Silico Analysis ◽  
High Binding Affinity ◽  
Cell Fusions

<p>The ongoing search to contain and control the spread of COVID-19 disease focuses on discovering drugs or vaccines that can play an essential role in treating this contagious disease. This paper focuses on natural compounds that can play a vital role in the treatment of Covid-19. The study spans over the chemicals that have the potential to bind with the key residues of type II Transmembrane Protease Serine (TMPRSS2). TMPRSS2 can be termed as the catalyst that cleaves the spike glycoproteins of Sars-Cov-2, which causes the replication and spread of virus inside the human body by facilitating virus-cell fusions. Drugs like Camostat Mesylate, Aprotinin, and Rimantadine have been proposed as potential inhibitors of TMPRSS2. After screening large sets of phytochemicals and flavonoids extracted from plants, potential compounds have been tested, and a set of most effective and suitable compounds are chosen for further studies. These selected compounds are further analyzed in terms of binding with key residues as well as high binding affinity with TMPRSS2. The in silico analysis of possible chemical compounds is carried out by using docking, screening analysis, Molecular Dynamics, and Electrostatic Potential Simulations. Chemicals extracted from different plants are comparatively analyzed with drugs like Aprotinin, Camostat Mesylate, and Rimantadine.</p>

scholarly journals Recombinant PhpA Protein, a Unique Histidine Motif-Containing Protein from Streptococcus pneumoniae, Protects Mice against Intranasal Pneumococcal Challenge

2001 ◽  
Vol 69 (6) ◽  
pp. 3827-3836 ◽  
Author(s):  
Ying Zhang ◽  
Amy W. Masi ◽  
Vicki Barniak ◽  
Ken Mountzouros ◽  
Margaret K. Hostetter ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Otitis Media ◽  
Conjugate Vaccine ◽  
Lipid A ◽  
Systemic Disease ◽  
Protein A ◽  
Complement C3 ◽  
Potential Candidate ◽  
Putative Open Reading Frame ◽  
Monophosphoryl Lipid A

ABSTRACT The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the current conjugate vaccine may still cause pneumococcal disease. To address these serotypes and the remaining otitis media due to Streptococcus pneumoniae, we have been evaluating antigenically conserved proteins from S. pneumoniae as vaccine candidates. A previous report identified a 20-kDa protein with putative human complement C3-proteolytic activity. By utilizing the publicly released pneumococcal genomic sequences, we found the gene encoding the 20-kDa protein to be part of a putative open reading frame of approximately 2,400 bp. We recombinantly expressed a 79-kDa fragment (rPhpA-79) that contains a repeated HxxHxH motif and evaluated it for vaccine potential. The antibodies elicited by the purified rPhpA-79 protein were cross-reactive to proteins from multiple strains of S. pneumoniae and were against surface-exposed epitopes. Immunization with rPhpA-79 protein adjuvanted with monophosphoryl lipid A (for subcutaneous immunization) or a mutant cholera toxin, CT-E29H (for intranasal immunization), protected CBA/N mice against death and bacteremia, as well as reduced nasopharyngeal colonization, following intranasal challenge with a heterologous pneumococcal strain. In contrast, immunization with the 20-kDa portion of the PhpA protein did not protect mice. These results suggest that rPhpA-79 is a potential candidate for use as a vaccine against pneumococcal systemic disease and otitis media.

scholarly journals Global Transcriptional Analysis of Dehydrated Salmonella enterica Serovar Typhimurium

2012 ◽  
Vol 78 (22) ◽  
pp. 7866-7875 ◽  
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.

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.

Changes in the composition and size distribution of endosperm proteins from bug-damaged wheats

2004 ◽  
Vol 55 (4) ◽  
pp. 477 ◽  
Author(s):  
D. Sivri ◽  
I. L. Batey ◽  
D. J. Skylas ◽  
L. Daqiq ◽  
C. W. Wrigley
Keyword(s):  
Size Distribution ◽  
High Performance ◽  
Control Sample ◽  
Size Exclusion ◽  
Endosperm Proteins ◽  
Sound Control ◽  
Glutenin Protein ◽  
And Control ◽  
Hmw Glutenins ◽  
Basic Region

In this study, grain that had been damaged by the bug Eurygaster spp. and/or Aelia spp., plus some undamaged grain, was selected from hard red winter (HRW) wheat. The changes in endosperm proteins were determined by 2-dimensional (2-D) electrophoresis and size-exclusion high-performance liquid chromatography (SE-HPLC). Although some new protein spots and a slight decrease in the staining intensities of some polypeptides were observed in the 2-D map of the bug-damaged sample, other parts of the gels were similar to the sound (control) sample in terms of relative mobilities and intensities of the polypeptide spots. The major difference between bug-damaged and control samples was that a group of polypeptides, presumably HMW-glutenins, shifted to a more basic region of the map. The SE-HPLC patterns of the total proteins extracted from control and bug-damaged samples in SDS-buffer showed that they differed in the size distribution of the polymeric glutenin protein and in their glutenin/gliadin ratios. The solubility of proteins in SDS buffer was greater in the bug-damaged sample. The 'unextractable' polymeric protein (only extractable in SDS-buffer after sonication) (UPP %) was significantly lower in the bug-damaged sample than in the control. The results of 2-D analysis and the decline in the quantity of unextractable proteins in SDS buffer suggest that bug-protease causes dough weakening by degradation of polymeric glutenin, presumably by hydrolysis, and possibly other mechanisms that affect the aggregation of the gluten molecules.

scholarly journals Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A

2020 ◽  
Vol 8 (6) ◽  
pp. 826
Author(s):  
Federica A. Falchi ◽  
Flaviana Di Lorenzo ◽  
Roberto Pizzoccheri ◽  
Gianluca Casino ◽  
Moira Paroni ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Lipid A ◽  
Cell Envelope ◽  
Osmotic Shock ◽  
Ectopic Expression ◽  
Phenotypic Effect ◽  
Osmotic Lysis ◽  
Membrane Defects ◽  
Inner Membrane Protein

LpxT is an inner membrane protein that transfers a phosphate group from the essential lipid undecaprenyl pyrophosphate (C-55PP) to the lipid A moiety of lipopolysaccharide, generating a lipid A tris-phosphorylated species. The protein is encoded by the non-essential lpxT gene, which is conserved in distantly related Gram-negative bacteria. In this work, we investigated the phenotypic effect of lpxT ectopic expression from a plasmid in Escherichia coli. We found that lpxT induction inhibited cell division and led to the formation of elongated cells, mostly with absent or altered septa. Moreover, the cells became sensitive to detergents and to hypo-osmotic shock, indicating that they had cell envelope defects. These effects were not due to lipid A hyperphosphorylation or C-55PP sequestering, but most likely to defective lipopolysaccharide transport. Indeed, lpxT overexpression in mutants lacking the L,D-transpeptidase LdtD and LdtE, which protect cells with outer membrane defects from osmotic lysis, caused cell envelope defects. Moreover, we found that pyrophosphorylated lipid A was also produced in a lpxT deletion mutant, indicating that LpxT is not the only protein able to perform such lipid A modification in E. coli.

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