scholarly journals Detection of Cytosolic Shigella flexneri via a C-Terminal Triple-Arginine Motif of GBP1 Inhibits Actin-Based Motility

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Anthony S. Piro ◽  
Dulcemaria Hernandez ◽  
Sarah Luoma ◽  
Eric M. Feeley ◽  
Ryan Finethy ◽  
...  
Keyword(s):  
Host Cell ◽  
Host Defense ◽  
Actin Polymerization ◽  
Bacterial Pathogens ◽  
Shigella Flexneri ◽  
Bacterial Species ◽  
Host Cells ◽  
Gram Negative Bacteria ◽  
Protein Motif ◽  
Gram Negative

ABSTRACT Dynamin-like guanylate binding proteins (GBPs) are gamma interferon (IFN-γ)-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol, but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique among the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria, Burkholderia thailandensis and Shigella flexneri. Rough lipopolysaccharide (LPS) mutants of S. flexneri colocalize with GBP1 less frequently than wild-type S. flexneri does, suggesting that host recognition of O antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C terminus, promotes the corecruitment of four additional GBP paralogs (GBP2, GBP3, GBP4, and GBP6). GBP1-decorated Shigella organisms replicate but fail to form actin tails, leading to their intracellular aggregation. Consequentially, the wild type but not the triple-arginine GBP1 mutant restricts S. flexneri cell-to-cell spread. Furthermore, human-adapted S. flexneri, through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis. These studies reveal that human GBP1 uniquely functions as an intracellular “glue trap,” inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense program, S. flexneri has evolved an effective counterdefense to restrict GBP1 recruitment. IMPORTANCE Several pathogenic bacterial species evolved to invade, reside in, and replicate inside the cytosol of their host cells. One adaptation common to most cytosolic bacterial pathogens is the ability to coopt the host’s actin polymerization machinery in order to generate force for intracellular movement. This actin-based motility enables Gram-negative bacteria, such as Shigella species, to propel themselves into neighboring cells, thereby spreading from host cell to host cell without exiting the intracellular environment. Here, we show that the human protein GBP1 acts as a cytosolic “glue trap,” capturing cytosolic Gram-negative bacteria through a unique protein motif and preventing disseminated infections in cell culture models. To escape from this GBP1-mediated host defense, Shigella employs a virulence factor that prevents or dislodges the association of GBP1 with cytosolic bacteria. Thus, therapeutic strategies to restore GBP1 binding to Shigella may lead to novel treatment options for shigellosis in the future. Several pathogenic bacterial species evolved to invade, reside in, and replicate inside the cytosol of their host cells. One adaptation common to most cytosolic bacterial pathogens is the ability to coopt the host’s actin polymerization machinery in order to generate force for intracellular movement. This actin-based motility enables Gram-negative bacteria, such as Shigella species, to propel themselves into neighboring cells, thereby spreading from host cell to host cell without exiting the intracellular environment. Here, we show that the human protein GBP1 acts as a cytosolic “glue trap,” capturing cytosolic Gram-negative bacteria through a unique protein motif and preventing disseminated infections in cell culture models. To escape from this GBP1-mediated host defense, Shigella employs a virulence factor that prevents or dislodges the association of GBP1 with cytosolic bacteria. Thus, therapeutic strategies to restore GBP1 binding to Shigella may lead to novel treatment options for shigellosis in the future.

scholarly journals Detection of cytosolicShigella flexnerivia a C-terminal triple-arginine motif of GBP1 inhibits actin-based motility

2017 ◽  
Author(s):  
Anthony S. Piro ◽  
Dulcemaria Hernandez ◽  
Sarah Luoma ◽  
Eric. M. Feeley ◽  
Ryan Finethy ◽  
...  
Keyword(s):  
Host Cell ◽  
Host Defense ◽  
Actin Polymerization ◽  
Bacterial Pathogens ◽  
Bacterial Species ◽  
Host Cells ◽  
Host Recognition ◽  
Gram Negative Bacteria ◽  
Defense Proteins ◽  
Gram Negative

AbstractDynamin-like guanylate binding proteins (GBPs) are gamma interferon (IFNγ)-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique amongst the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria,Burkholderia thailandensisandShigella flexneri.Rough lipopolysaccharide (LPS) mutants ofS. flexnerico-localize with GBP1 less frequently than wildtypeS. flexneri, suggesting that host recognition of O-antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C-terminus, promotes the co-recruitment of four additional GBP paralogs (GBP2, GBP3, GBP4 and GBP6). GBP1-decoratedShigellareplicate but fail to form actin tails leading to their intracellular aggregation. Consequentially, wildtype but not the triple-arginine GBP1 mutant restrictsS. flexnericell-to-cell spread. Furthermore, human-adaptedS. flexneri,through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillusB. thailandensis. These studies reveal that human GBP1 uniquely functions as an intracellular ‘glue trap’ inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense programS. flexnerihas evolved an effective counter-defense to restrict GBP1 recruitment.ImportanceSeveral pathogenic bacterial species evolved to invade, reside and replicate inside the cytosol of their host cells. One adaptation common to most cytosolic bacterial pathogens is the ability to co-opt the host’s actin polymerization machinery, in order to generate force for intracellular movement. This actin-based motility enables Gram-negative bacteria such asShigellato propel themselves into neighboring cells thereby spreading from host cell to host cell without exiting the intracellular environment. Here, we show that the human protein GBP1 acts as a cytosolic ‘glue trap’ capturing cytosolic Gram-negative bacteria through a unique protein motif and preventing disseminated infections in cell culture models. To escape from this GBP1-mediated host defense,Shigellaemploys a virulence factor that prevents or dislodges the association of GBP1 with cytosolic bacteria. Thus, therapeutic strategies to restore GBP1 binding toShigellamay lead to novel treatment options for shigellosis in the future.

scholarly journals The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Lihi Shaulov ◽  
Jenia Gershberg ◽  
Wanyin Deng ◽  
B. Brett Finlay ◽  
Neta Sal-Man
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Bacterial Pathogens ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Bacterial Virulence ◽  
Effector Proteins ◽  
Host Cells ◽  
Gram Negative ◽  
Type Iii

ABSTRACT The type III secretion system (T3SS) is a multiprotein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. To ensure that effector proteins are efficiently translocated into the host cell, bacteria must be able to sense their contact with the host cell. In this study, we found that EscP, which was previously shown to function as the ruler protein of the enteropathogenic Escherichia coli T3SS, is also involved in the switch from the secretion of translocator proteins to the secretion of effector proteins. In addition, we demonstrated that EscP can interact with the gatekeeper protein SepL and that the EscP-SepL complex dissociates upon a calcium concentration drop. We suggest a model in which bacterial contact with the host cell is accompanied by a drop in the calcium concentration that causes SepL-EscP complex dissociation and triggers the secretion of effector proteins. IMPORTANCE The emergence of multidrug-resistant bacterial strains, especially those of pathogenic bacteria, has serious medical and clinical implications. At the same time, the development and approval of new antibiotics have been limited for years. Recently, antivirulence drugs have received considerable attention as a novel antibiotic strategy that specifically targets bacterial virulence rather than growth, an approach that applies milder evolutionary pressure on the bacteria to develop resistance. A highly attractive target for the development of antivirulence compounds is the type III secretion system, a specialized secretory system possessed by many Gram-negative bacterial pathogens for injecting virulence factors (effectors) into host cells. In this study, we shed light on the molecular mechanism that allows bacteria to sense their contact with the host cell and to respond with the timed secretion of effector proteins. Understanding this critical step for bacterial virulence may provide a new therapeutic strategy. IMPORTANCE The emergence of multidrug-resistant bacterial strains, especially those of pathogenic bacteria, has serious medical and clinical implications. At the same time, the development and approval of new antibiotics have been limited for years. Recently, antivirulence drugs have received considerable attention as a novel antibiotic strategy that specifically targets bacterial virulence rather than growth, an approach that applies milder evolutionary pressure on the bacteria to develop resistance. A highly attractive target for the development of antivirulence compounds is the type III secretion system, a specialized secretory system possessed by many Gram-negative bacterial pathogens for injecting virulence factors (effectors) into host cells. In this study, we shed light on the molecular mechanism that allows bacteria to sense their contact with the host cell and to respond with the timed secretion of effector proteins. Understanding this critical step for bacterial virulence may provide a new therapeutic strategy.

Ultrastructure of periplasmic bodies in gram-negative bacteria

1990 ◽  
Vol 48 (3) ◽  
pp. 640-641
Author(s):  
Xie Nianming ◽  
Ding Shaoqing ◽  
Wang Luping ◽  
Yuan Zenglin ◽  
Zhan Guolai ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Campylobacter Jejuni ◽  
Proteus Mirabilis ◽  
Shigella Flexneri ◽  
Morphological Description ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Clostridium Tetani ◽  
Gram Negative ◽  
Brucella Canis

Perhaps the data about periplasmic enzymes are obtained through biochemical methods but lack of morphological description. We have proved the existence of periplasmic bodies by electron microscope and described their ultrastructures. We hope this report may draw the attention of biochemists and mrophologists to collaborate on researches in periplasmic enzymes or periplasmic bodies with each other.One or more independent bodies may be seen in the periplasmic space between outer and inner membranes of Gram-negative bacteria, which we called periplasmic bodies. The periplasmic bodies have been found in seven species of bacteria at least, including the Pseudomonas aeroginosa. Shigella flexneri, Echerichia coli. Yersinia pestis, Campylobacter jejuni, Proteus mirabilis, Clostridium tetani. Vibrio cholerae and Brucella canis.

scholarly journals Role of Autocleavage in the Function of a Type III Secretion Specificity Switch Protein in Salmonella enterica Serovar Typhimurium

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Julia V. Monjarás Feria ◽  
Matthew D. Lefebre ◽  
York-Dieter Stierhof ◽  
Jorge E. Galán ◽  
Samuel Wagner
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Effector Proteins ◽  
Switching Function ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative ◽  
Type Iii ◽  
Autocatalytic Cleavage ◽  
Serovar Typhimurium

ABSTRACTType III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the “switch protein,” a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of theSalmonella entericaserovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage eventper sedoes not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function.IMPORTANCEBacterial interaction with eukaryotic hosts often involves complex molecular machines for targeted delivery of bacterial effector proteins. One such machine, the type III secretion system of some Gram-negative bacteria, serves to inject a multitude of structurally diverse bacterial proteins into the host cell. Critical to the function of these systems is their ability to secrete proteins in a strict hierarchical order, but it is unclear how the mechanism of switching works. Central to the switching mechanism is a highly conserved inner membrane protease that undergoes autocatalytic cleavage. Although it has been suggested previously that the autocleavage event is the trigger for substrate switching, we show here that this is not the case. Rather, our results show that cleavage allows the proper conformation of the protein to render it competent for its switching function. These findings may help develop inhibitors of type III secretion machines that offer novel therapeutic avenues to treat various infectious diseases.

scholarly journals In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A

2018 ◽  
Vol 10 (464) ◽  
pp. eaal0033 ◽  
Author(s):  
Ahsan R. Akram ◽  
Sunay V. Chankeshwara ◽  
Emma Scholefield ◽  
Tashfeen Aslam ◽  
Neil McDonald ◽  
...  
Keyword(s):  
Lipid A ◽  
Respiratory Infections ◽  
Bacterial Species ◽  
Water Soluble ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Human Lungs ◽  
Fluorescent Peptide ◽  
Bacterial Lipid

Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time. We used optical endomicroscopy to visualize a water-soluble optical imaging probe based on the antimicrobial peptide polymyxin conjugated to an environmentally sensitive fluorophore. The probe was chemically stable and nontoxic and, after in-human intrapulmonary microdosing, enabled the specific detection of Gram-negative bacteria in distal human airways and alveoli within minutes. The results suggest that pulmonary molecular imaging using a topically administered fluorescent probe targeting bacterial lipid A is safe and practical, enabling rapid in situ identification of Gram-negative bacteria in humans.

scholarly journals Antimicrobial-Resistant Enteric Bacteria from Dairy Cattle

2006 ◽  
Vol 73 (1) ◽  
pp. 156-163 ◽  
Author(s):  
Ashish A. Sawant ◽  
Narasimha V. Hegde ◽  
Beth A. Straley ◽  
Sarah C. Donaldson ◽  
Brenda C. Love ◽  
...  
Keyword(s):  
Dairy Cattle ◽  
Dna Hybridization ◽  
Bacterial Species ◽  
Dairy Herd ◽  
Enteric Bacteria ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Predominant Species ◽  
Lactating Dairy Cattle

ABSTRACT A study was conducted to understand the descriptive and molecular epidemiology of antimicrobial-resistant gram-negative enteric bacteria in the feces of healthy lactating dairy cattle. Gram-negative enteric bacteria resistant to ampicillin, florfenicol, spectinomycin, and tetracycline were isolated from the feces of 35, 8, 5, and 42% of 213 lactating cattle on 74, 39, 9, 26, and 82% of 23 farms surveyed, respectively. Antimicrobial-resistant gram-negative bacteria accounted for 5 (florfenicol) to 14% (tetracycline) of total gram-negative enteric microflora. Nine bacterial species were isolated, of which Escherichia coli (87%) was the most predominant species. MICs showing reduced susceptibility to ampicillin, ceftiofur, chloramphenicol, florfenicol, spectinomycin, streptomycin, and tetracycline were observed in E. coli isolates. Isolates exhibited resistance to ampicillin (48%), ceftiofur (11%), chloramphenicol (20%), florfenicol (78%), spectinomycin (18%), and tetracycline (93%). Multidrug resistance (≥3 to 6 antimicrobials) was seen in 40% of E. coli isolates from healthy lactating cattle. Of 113 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 93% of isolates, while the remaining 7% isolates carried the tet(A) determinant. DNA-DNA hybridization assays revealed that tet determinants were located on the chromosome. Pulsed-field gel electrophoresis revealed that tetracycline-resistant E. coli isolates (n = 99 isolates) belonged to 60 subtypes, which is suggestive of a highly diverse population of tetracycline-resistant organisms. On most occasions, E. coli subtypes, although shared between cows within the herd, were confined mostly to a dairy herd. The findings of this study suggest that commensal enteric E. coli from healthy lactating cattle can be an important reservoir for tetracycline and perhaps other antimicrobial resistance determinants.

scholarly journals Evolutionary Perspectives on the Moonlighting Functions of Bacterial Factors That Support Actin-Based Motility

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Volkan K. Köseoğlu ◽  
Hervé Agaisse
Keyword(s):  
Cell Adhesion ◽  
Host Cell ◽  
Biofilm Formation ◽  
Actin Polymerization ◽  
Current Knowledge ◽  
Bacterial Pathogens ◽  
Actin Nucleation ◽  
Bacterial Aggregation ◽  
Moonlighting Functions ◽  
Evolutionary Perspectives

ABSTRACT Various bacterial pathogens display an intracellular lifestyle and spread from cell to cell through actin-based motility (ABM). ABM requires actin polymerization at the bacterial pole and is mediated by the expression of bacterial factors that hijack the host cell actin nucleation machinery or exhibit intrinsic actin nucleation properties. It is increasingly recognized that bacterial ABM factors, in addition to having a crucial task during the intracellular phase of infection, display “moonlighting” adhesin functions, such as bacterial aggregation, biofilm formation, and host cell adhesion/invasion. Here, we review our current knowledge of ABM factors and their additional functions, and we propose that intracellular ABM functions have evolved from ancestral, extracellular adhesin functions.

scholarly journals A new form of actin assembly around the Shigella-containing vacuole regulates its intracellular niche

2020 ◽  
Author(s):  
Sonja Kühn ◽  
John Bergqvist ◽  
Laura Barrio ◽  
Stephanie Lebreton ◽  
Chiara Zurzolo ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
De Novo ◽  
Shigella Flexneri ◽  
Host Cells ◽  
Host Recognition ◽  
Actin Assembly ◽  
Actin Structure ◽  
New Form ◽  
Structure And Functions

SUMMARYThe enteroinvasive bacterium Shigella flexneri forces its uptake into non-phagocytic host cells through the translocation of T3SS effectors that subvert the actin cytoskeleton. Here, we report de novo actin polymerization after cellular entry around the bacterial containing vacuole (BCV) leading to the formation of a dynamic actin cocoon. This cocoon is thicker than any described cellular actin structure and functions as a gatekeeper for the cytosolic access of the pathogen. Host Cdc42, Toca-1, N-WASP, WIP, the Arp2/3 complex, cortactin, coronin, and cofilin are recruited to the actin cocoon. They are subverted by T3SS effectors, such as IpgD, IpgB1, and IcsB. IcsB immobilizes components of the actin polymerization machinery at the BCV. This represents a novel microbial subversion strategy through localized entrapment of host actin regulators causing massive actin assembly. We propose that the cocoon protects Shigella’s niche from canonical maturation or host recognition.

scholarly journals Structural insights into a novel family of integral membrane siderophore reductases

2021 ◽  
Vol 118 (34) ◽  
pp. e2101952118
Author(s):  
Inokentijs Josts ◽  
Katharina Veith ◽  
Vincent Normant ◽  
Isabelle J. Schalk ◽  
Henning Tidow
Keyword(s):  
Iron Uptake ◽  
Bacterial Species ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Inner Membrane Protein ◽  
Uptake Studies ◽  
Structural Insights

Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from Pseudomonas aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a fold with two tightly bound heme molecules. In combination with in vitro reduction assays and in vivo iron uptake studies, these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.

scholarly journals Lysis Genes of the Bacillus subtilisDefective Prophage PBSX

1998 ◽  
Vol 180 (8) ◽  
pp. 2110-2117 ◽  
Author(s):  
Susanne Krogh ◽  
Steen T. Jørgensen ◽  
Kevin M. Devine
Keyword(s):  
Functional Analysis ◽  
Host Cell ◽  
Expression System ◽  
Cell Lysis ◽  
Lethal Gene ◽  
Gram Negative Bacteria ◽  
Gene Products ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Exported Protein

ABSTRACT Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they arexepA, encoding an exported protein; xhlA, encoding a putative membrane-associated protein; xhlB, encoding a putative holin; and xlyA, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of Bacillus subtilis 168. The results show thatxepA is unlikely to be involved in host cell lysis. Expression of both xhlA and xhlB is necessary to effect host cell lysis of B. subtilis. Expression ofxhlB (encoding the putative holin) together withxlyA (encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when xlyA is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.

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