scholarly journals Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence

2021 ◽  
Author(s):  
Mathias Diehl ◽  
Sebastian Weber ◽  
Marek Cyrklaff ◽  
Cecilia P. Sanchez ◽  
Carlo A. Beretta ◽  
...  
Keyword(s):  
Host Cell ◽  
Wild Type ◽  
Wild Type Phenotype ◽  
Exact Function ◽  
Independent Mechanism ◽  
Host Origin ◽  
Total Lack ◽  
J Domain ◽  
Morphology Of Surface ◽  
Human Hsp70

AbstractThe pathology associated with malaria infection is largely due to the ability of infected human erythrocytes to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin.Taken together, our data reveal a role for PFA66 in host cell modification, implicate human HSP70 as also being essential in this process, and uncover a KAHRP-independent mechanism for correct knob biogenesis. Our observations open up exciting new avenues for the development of new anti-malarials.

scholarly journals Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence

2021 ◽  
Vol 17 (10) ◽  
pp. e1009969
Author(s):  
Mathias Diehl ◽  
Lena Roling ◽  
Lukas Rohland ◽  
Sebastian Weber ◽  
Marek Cyrklaff ◽  
...  
Keyword(s):  
Host Cell ◽  
Wild Type ◽  
Functional Interaction ◽  
Wild Type Phenotype ◽  
Exact Function ◽  
Host Origin ◽  
Total Lack ◽  
J Domain ◽  
Human Rbcs ◽  
Morphology Of Surface

The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis.

scholarly journals Mycobacterium bovis Requires P27 (LprG) To Arrest Phagosome Maturation and Replicate within Bovine Macrophages

2016 ◽  
Vol 85 (3) ◽  
Author(s):  
Cristina Lourdes Vázquez ◽  
María Verónica Bianco ◽  
Federico Carlos Blanco ◽  
Marina Andrea Forrellad ◽  
Maximiliano Gabriel Gutierrez ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Mutant Strain ◽  
Mycobacterium Bovis ◽  
Phagosome Maturation ◽  
Wild Type ◽  
Content Type ◽  
Bovine Macrophage ◽  
Wild Type Phenotype ◽  
First Time

ABSTRACT Mycobacterium bovis causes tuberculosis in a wide variety of mammals, with strong tropism for cattle and eventually humans. P27, also called LprG, is among the proteins involved in the mechanisms of the virulence and persistence of M. bovis and Mycobacterium tuberculosis. Here, we describe a novel function of P27 in the interaction of M. bovis with its natural host cell, the bovine macrophage. We found that a deletion in the p27-p55 operon impairs the replication of M. bovis in bovine macrophages. Importantly, we show for the first time that M. bovis arrests phagosome maturation in a process that depends on P27. This effect is P27 specific since complementation with wild-type p27 but not p55 fully restored the wild-type phenotype of the mutant strain; this indicates that P55 plays no important role during the early events of M. bovis infection. In addition, we also showed that the presence of P27 from M. smegmatis decreases the association of LAMP-3 with bead phagosomes, indicating that P27 itself blocks phagosome-lysosome fusion by modulating the traffic machinery in the cell host.

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 Ehrlichia chaffeensis and Its Invasin EtpE Block Reactive Oxygen Species Generation by Macrophages in a DNase X-Dependent Manner

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Omid Teymournejad ◽  
Mingqun Lin ◽  
Yasuko Rikihisa
Keyword(s):  
Host Cell ◽  
Cell Receptor ◽  
Ros Generation ◽  
Ehrlichia Chaffeensis ◽  
Biological Processes ◽  
Wild Type ◽  
New Approach ◽  
Host Cell Receptor ◽  
Life Threatening ◽  
Human Monocytic Ehrlichiosis

ABSTRACT The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most genes that confer resistance to oxidative stress but can block reactive oxygen species (ROS) generation by host monocytes-macrophages. Bacterial and host molecules responsible for this inhibition have not been identified. To infect host cells, Ehrlichia uses the C terminus of its surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C), which directly binds the mammalian cell surface receptor glycosylphosphatidylinositol-anchored protein DNase X. We investigated whether EtpE-C binding to DNase X blocks ROS production by mouse bone marrow-derived macrophages (BMDMs). On the basis of a luminol-dependent chemiluminescence assay, E. chaffeensis inhibited phorbol myristate acetate (PMA)-induced ROS generation by BMDMs from wild-type, but not DNase X−/−, mice. EtpE-C is critical for inhibition, as recombinant EtpE-C (rEtpE-C)-coated latex beads, but not recombinant N-terminal EtpE-coated or uncoated beads, inhibited PMA-induced ROS generation by BMDMs from wild-type mice. DNase X is required for this inhibition, as none of these beads inhibited PMA-induced ROS generation by BMDMs from DNase X−/− mice. Previous studies showed that E. chaffeensis does not block ROS generation in neutrophils, a cell type that is a potent ROS generator but is not infected by E. chaffeensis. Human and mouse peripheral blood neutrophils did not express DNase X. Our findings point to a unique survival mechanism of ROS-sensitive obligate intramonocytic bacteria that involves invasin EtpE binding to DNase X on the host cell surface. This is the first report of bacterial invasin having such a subversive activity on ROS generation. IMPORTANCE Ehrlichia chaffeensis preferentially infects monocytes-macrophages and causes a life-threatening emerging tick-transmitted infectious disease called human monocytic ehrlichiosis. Ehrlichial infection, and hence the disease, depends on the ability of this bacterium to avoid or overcome powerful microbicidal mechanisms of host monocytes-macrophages, one of which is the generation of ROS. Our findings reveal that an ehrlichial surface invasin, EtpE, not only triggers bacterial entry but also blocks ROS generation by host macrophages through its host cell receptor, DNase X. As ROS sensitivity is an Achilles’ heel of this group of pathogens, understanding the mechanism by which E. chaffeensis rapidly blocks ROS generation suggests a new approach for developing effective anti-infective measures. The discovery of a ROS-blocking pathway is also important, as modulation of ROS generation is important in a variety of ailments and biological processes. IMPORTANCE Ehrlichia chaffeensis preferentially infects monocytes-macrophages and causes a life-threatening emerging tick-transmitted infectious disease called human monocytic ehrlichiosis. Ehrlichial infection, and hence the disease, depends on the ability of this bacterium to avoid or overcome powerful microbicidal mechanisms of host monocytes-macrophages, one of which is the generation of ROS. Our findings reveal that an ehrlichial surface invasin, EtpE, not only triggers bacterial entry but also blocks ROS generation by host macrophages through its host cell receptor, DNase X. As ROS sensitivity is an Achilles’ heel of this group of pathogens, understanding the mechanism by which E. chaffeensis rapidly blocks ROS generation suggests a new approach for developing effective anti-infective measures. The discovery of a ROS-blocking pathway is also important, as modulation of ROS generation is important in a variety of ailments and biological processes.

scholarly journals The UDP N-Acetylgalactosamine 4-Epimerase Gene Is Essential for Mesophilic Aeromonas hydrophila Serotype O34 Virulence

2006 ◽  
Vol 74 (1) ◽  
pp. 537-548 ◽  
Author(s):  
Rocío Canals ◽  
Natalia Jiménez ◽  
Silvia Vilches ◽  
Miguel Regué ◽  
Susana Merino ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sugar Residue ◽  
Serum Resistance ◽  
Single Mutation ◽  
Wild Type ◽  
O Antigen ◽  
Wild Type Phenotype

ABSTRACT Mesophilic Aeromonas hydrophila strains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP N-acetylgalactosamine 4-epimerase (gne) confers the O− phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The gne gene is present in all the mesophilic Aeromonas strains tested. No changes were observed for the LPS core in a gne mutant from A. hydrophila strain AH-3 (serotype O34). O34 antigen LPS contains N-acetylgalactosamine, while no such sugar residue forms part of the LPS core from A. hydrophila AH-3. Some of the pathogenic features of A. hydrophila AH-3 gne mutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the gne mutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic Aeromonas strains, possess two kinds of flagella, and the absence of O34 antigen molecules by gne mutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type gne gene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).

Rescue of the mutant phenotype by reexpression of full-length vinculin in null F9 cells; effects on cell locomotion by domain deleted vinculin

1998 ◽  
Vol 111 (11) ◽  
pp. 1535-1544 ◽  
Author(s):  
W. Xu ◽  
J.L. Coll ◽  
E.D. Adamson
Keyword(s):  
Marked Effect ◽  
Control Cell ◽  
Covalent Attachment ◽  
Tail Domain ◽  
Wild Type ◽  
F9 Cells ◽  
Wild Type Phenotype ◽  
Correct Location ◽  
Low Levels ◽  
Lamellipodia Formation

Vinculin plays a role in signaling between integrins and the actin cytoskeleton. We reported earlier that F9-derived cells lacking vinculin are less spread, less adhesive, and move two times faster than wild-type F9 cells. Expression of intact vinculin in null cells restored all wild-type characteristics. In contrast, expression of the head (90 kDa) fragment exaggerated mutant characteristics, especially locomotion, which was double that of vinculin null cells. Expression of the tail domain also had a marked effect on locomotion in the opposite direction, reducing it to very low levels. The expression of the head plus tail domains together (no covalent attachment) effected a partial rescue towards wild-type phenotype, thus indicating that reexpressed polypeptides may be in their correct location and are interacting normally. Therefore, we conclude that: (1) the head domain is part of the locomotory force of the cell, modulated by the tail, and driven by the integrin/matrix connection; (2) intact vinculin is required for normal regulation of cell behavior, suggesting that vinculin head-tail interactions control cell adhesion, spreading, lamellipodia formation and locomotion.

Enteropathogenic Escherichia coli adherence to intestinal epithelial monolayers diminishes barrier function

1995 ◽  
Vol 268 (2) ◽  
pp. G374-G379 ◽  
Author(s):  
J. Spitz ◽  
R. Yuhan ◽  
A. Koutsouris ◽  
C. Blatt ◽  
J. Alverdy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Barrier Function ◽  
Cell Function ◽  
Cytoskeletal Proteins ◽  
Calcium Stores ◽  
Intestinal Epithelial ◽  
Enteropathogenic Escherichia Coli ◽  
Wild Type ◽  
Cell Monolayers

The mechanism by which enteropathogenic Escherichia coli (EPEC) causes diarrhea remains elusive. Several alterations within the host cell have been demonstrated to occur following EPEC attachment including increases in intracellular Ca2+ concentration and rearrangement and phosphorylation of several cytoskeletal proteins. The consequences of these intracellular perturbations on host cell function, however, have not been determined. The aim of this study was to examine the effect of EPEC adherence on intestinal epithelial barrier function. T84 cell monolayers were infected with either wild-type EPEC or a nonadherent isogenic derivative. Transepithelial electrical resistance, a measure of barrier function, decreased 33.5 +/- 6.4% after a 6-h incubation with the wild-type strain. Electron microscopy revealed ultrastructurally normal cells, and lactate dehydrogenase release assays failed to demonstrate cytotoxicity. Dual 22Na+ and [3H]mannitol flux studies localized the permeability defect to tight junctions. In addition, cumulative flux of the paracellular marker mannitol was four- to fivefold greater across monolayers infected with wild-type EPEC. Sequestration of intracellular calcium stores by dantrolene completely abrogated the resistance drop associated with EPEC attachment. These data demonstrate that adherence of EPEC to intestinal epithelial cell monolayers disrupts tight junction barrier function via a calcium-requiring event.

scholarly journals New Sporulation Loci in Streptomyces coelicolor A3(2)

1999 ◽  
Vol 181 (17) ◽  
pp. 5419-5425 ◽  
Author(s):  
N. Jamie Ryding ◽  
Maureen J. Bibb ◽  
Virginie Molle ◽  
Kim C. Findlay ◽  
Keith F. Chater ◽  
...  
Keyword(s):  
Phase Contrast ◽  
Streptomyces Coelicolor ◽  
Cosmid Library ◽  
Phase Contrast Microscopy ◽  
Wild Type ◽  
Chromosomal Dna ◽  
East Anglia ◽  
Wild Type Phenotype ◽  
Contrast Microscopy ◽  
Spore Pigment

ABSTRACT Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the grey polyketide spore pigment, and such white (whi) mutants had been used to define eight sporulation loci, whiA,whiB, whiD, whiE, whiG,whiH, whiI, and whiJ (K. F. Chater, J. Gen. Microbiol. 72:9–28, 1972; N. J. Ryding, Ph.D. thesis, University of East Anglia, 1995). In an attempt to identify new whi loci, we mutagenized S. coelicolor M145 spores with nitrosoguanidine and identified 770 mutants with colonies ranging from white to medium grey. After excluding unstable strains, we examined the isolates by phase-contrast microscopy and chose 115 whi mutants with clear morphological phenotypes for further study. To exclude mutants representing cloned whi genes, self-transmissible SCP2*-derived plasmids carrying whiA, whiB,whiG, whiH, or whiJ (but notwhiD, whiE, or whiI) were introduced into each mutant by conjugation, and strains in which the wild-type phenotype was restored either partially or completely by any of these plasmids were excluded from further analysis. In an attempt to complement some of the remaining 31 whi mutants, an SCP2* library of wild-type S. coelicolor chromosomal DNA was introduced into 19 of the mutants by conjugation. Clones restoring the wild-type phenotype to 12 of the 19 strains were isolated and found to represent five distinct loci, designated whiK,whiL, whiM, whiN, andwhiO. Each of the five loci was located on the ordered cosmid library: whiL, whiM, whiN, and whiO occupied positions distinct from previously clonedwhi genes; whiK was located on the same cosmid overlap as whiD, but the two loci were shown by complementation to be distinct. The phenotypes resulting from mutations at each of these new loci are described.

scholarly journals The Helicobacter pylori Autotransporter ImaA Tempers the Bacterium's Interaction with α5β1 Integrin

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
William E. Sause ◽  
Daniela Keilberg ◽  
Soufiane Aboulhouda ◽  
Karen M. Ottemann
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Wild Type ◽  
Content Type ◽  
Type Iv ◽  
H Pylori ◽  
Α5β1 Integrin ◽  
Cell Interface

ABSTRACT The human pathogen Helicobacter pylori uses the host receptor α5β1 integrin to trigger inflammation in host cells via its cag pathogenicity island (cag PAI) type IV secretion system (T4SS). Here, we report that the H. pylori ImaA protein (HP0289) decreases the action of the cag PAI T4SS via tempering the bacterium's interaction with α5β1 integrin. Previously, imaA-null mutants were found to induce an elevated inflammatory response that was dependent on the cag PAI T4SS; here we extend those findings to show that the elevated response is independent of the CagA effector protein. To understand how ImaA could be affecting cag PAI T4SS activity at the host cell interface, we utilized the Phyre structural threading program and found that ImaA has a region with remote homology to bacterial integrin-binding proteins. This region was required for ImaA function. Unexpectedly, we observed that imaA mutants bound higher levels of α5β1 integrin than wild-type H. pylori, an outcome that required the predicted integrin-binding homology region of ImaA. Lastly, we report that ImaA directly affected the amount of host cell β1 integrin but not other cellular integrins. Our results thus suggest a model in which H. pylori employs ImaA to regulate interactions between integrin and the T4SS and thus alter the host inflammatory strength.

scholarly journals Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Susmita Ghosh ◽  
Elizabeth A. Ruelke ◽  
Joshua C. Ferrell ◽  
Maria D. Bodero ◽  
Kenneth A. Fields ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Host Cells ◽  
Actin Binding ◽  
Wild Type ◽  
Content Type ◽  
Allelic Exchange ◽  
Binding Domains

ABSTRACT The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

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