scholarly journals The Sca2 Autotransporter Protein from Rickettsia conorii Is Sufficient To Mediate Adherence to and Invasion of Cultured Mammalian Cells

2009 ◽  
Vol 77 (12) ◽  
pp. 5272-5280 ◽  
Author(s):  
Marissa M. Cardwell ◽  
Juan J. Martinez
Keyword(s):  
Mammalian Cells ◽  
Spotted Fever ◽  
Critical Role ◽  
Bioinformatic Analysis ◽  
Surface Proteins ◽  
Host Cells ◽  
Mammalian Host ◽  
Rickettsia Conorii ◽  
Spotted Fever Group ◽  
E Coli

ABSTRACT Obligate intracellular bacteria of the genus Rickettsia must adhere to and invade the host endothelium in order to establish an infection. These processes require the interaction of rickettsial surface proteins with mammalian host cell receptors. A previous bioinformatic analysis of sequenced rickettsial species identified a family of at least 17 predicted “surface cell antigen” (sca) genes whose products resemble autotransporter proteins. Two members of this family, rOmpA and rOmpB of spotted fever group (SFG) rickettsiae have been identified as adhesion and invasion factors, respectively; however, little is known about the putative functions of the other sca gene products. An intact sca2 gene is found in the majority of pathogenic SFG rickettsiae and, due to its sequence conservation among these species, we predict that Sca2 may play an important function at the rickettsial surface. Here we have shown that sca2 is transcribed and expressed in Rickettsia conorii and have used a heterologous gain-of-function assay in E. coli to determine the putative role of Sca2. Using this system, we have demonstrated that expression of Sca2 at the outer membrane of nonadherent, noninvasive E. coli is sufficient to mediate adherence to and invasion of a panel of mammalian cells, including endothelial cells. Furthermore, soluble Sca2 protein is capable of diminishing R. conorii invasion of cultured mammalian cells. This is the first evidence that Sca2 participates in the interaction between SFG rickettsiae and host cells and suggests that in addition to other surface proteins, Sca2 may play a critical role in rickettsial pathogenesis.

scholarly journals The Rickettsia conorii Autotransporter Protein Sca1 Promotes Adherence to Nonphagocytic Mammalian Cells

2010 ◽  
Vol 78 (5) ◽  
pp. 1895-1904 ◽  
Author(s):  
Sean P. Riley ◽  
Kenneth C. Goh ◽  
Timothy M. Hermanas ◽  
Marissa M. Cardwell ◽  
Yvonne G. Y. Chan ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Spotted Fever ◽  
Expression System ◽  
Host Cells ◽  
Rickettsia Conorii ◽  
Spotted Fever Group ◽  
Gram Negative Bacteria ◽  
Heterologous Expression System ◽  
Cell Association ◽  
Sfg Rickettsia

ABSTRACT The pathogenesis of spotted fever group (SFG) Rickettsia species, including R. conorii and R. rickettsii, is acutely dependent on adherence to and invasion of host cells, including cells of the mammalian endothelial system. Bioinformatic analyses of several rickettsia genomes revealed the presence of a cohort of genes designated sca genes that are predicted to encode proteins with homology to autotransporter proteins of Gram-negative bacteria. Previous work demonstrated that three members of this family, rOmpA (Sca0), Sca2, and rOmpB (Sca5) are involved in the interaction with mammalian cells; however, very little was known about the function of other conserved rickettsial Sca proteins. Here we demonstrate that sca1, a gene present in nearly all SFG rickettsia genomes, is actively transcribed and expressed in R. conorii cells. Alignment of Sca1 sequences from geographically diverse SFG Rickettsia species showed that there are high degrees of sequence identity and conservation of these sequences, suggesting that Sca1 may have a conserved function. Using a heterologous expression system, we demonstrated that production of R. conorii Sca1 in the Escherichia coli outer membrane is sufficient to mediate attachment to but not invasion of a panel of cultured mammalian epithelial and endothelial cells. Furthermore, preincubation of a recombinant Sca1 peptide with host cells blocked R. conorii cell association. Together, these results demonstrate that attachment to mammalian cells can be uncoupled from the entry process and that Sca1 is involved in the adherence of R. conorii to host cells.

Movement of spotted fever rickettsiae through tick host cells in vitro

1996 ◽  
Vol 54 ◽  
pp. 810-811
Author(s):  
U. G. Munderloh ◽  
S. F. Hayes ◽  
J. Cummings ◽  
T. J. Kurtti
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Spotted Fever ◽  
Shigella Flexneri ◽  
Light And Electron Microscopy ◽  
Host Cells ◽  
Spotted Fever Group ◽  
Symbiotic Association ◽  
Obligate Intracellular

Spotted fever group (SFG) rickettsiae are obligate intracellular prokaryotes that include tick-borne pathogens of animals and man as well as organisms that live in symbiotic association with their tick hosts. A striking feature of the behavior of pathogenic rickettsiae in the vertebrate is their ability to quickly disseminate between cells from the original site of entry shortly after infection, and before severe lesions are detected. Similarly, ticks become systemically infected with SFG rickettsiae, indicating that an efficient mechanism of dispersal also exists in the vector. This is accomplished despite the fact that rickettsiae are not motile.Kadurugamuwa et al. (1991) have used light and electron microscopy to show that Shigella flexneri utilize host cytoskeletal components to travel through cytoplasmic extensions and penetrate into neighboring cells. Using mammalian cells cultured in vitro, Heinzen et al. (1993) have demonstrated that SFG rickettsiae cause host cell actin polymerization at one rickettsial pole causing them to be propelled through the cytoplasm, and to transfer rapidly from cell to cell.

Evolution, purification, and characterization of RC0497: a peptidoglycan amidase from the prototypical spotted fever species Rickettsia conorii

2020 ◽  
Vol 401 (2) ◽  
pp. 249-262 ◽  
Author(s):  
Jignesh G. Patel ◽  
Hema P. Narra ◽  
Krishna Mohan Sepuru ◽  
Abha Sahni ◽  
Sandhya R. Golla ◽  
...  
Keyword(s):  
Cell Wall ◽  
Spotted Fever ◽  
Protein A ◽  
Rickettsia Conorii ◽  
Spotted Fever Group ◽  
Purification And Characterization ◽  
E Coli ◽  
Binding Domains ◽  
Α Helix

AbstractRickettsial species have independently lost several genes owing to reductive evolution while retaining those predominantly implicated in virulence, survival, and biosynthetic pathways. In this study, we have identified a previously uncharacterized Rickettsia conorii gene RC0497 as an N-acetylmuramoyl-L-alanine amidase constitutively expressed during infection of cultured human microvascular endothelial cells at the levels of both mRNA transcript and encoded protein. A homology-based search of rickettsial genomes reveals that RC0497 homologs, containing amidase_2 family and peptidoglycan binding domains, are highly conserved among the spotted fever group (SFG) rickettsiae. The recombinant RC0497 protein exhibits α-helix secondary structure, undergoes a conformational change in the presence of zinc, and exists as a dimer at higher concentrations. We have further ascertained the enzymatic activity of RC0497 via demonstration of its ability to hydrolyze Escherichia coli peptidoglycan. Confocal microscopy on E. coli expressing RC0497 and transmission immunoelectron microscopy of R. conorii revealed its localization predominantly to the cell wall, septal regions of replicating bacteria, and the membrane of vesicles pinching off the cell wall. In summary, we have identified and functionally characterized RC0497 as a peptidoglycan hydrolase unique to spotted fever rickettsiae, which may potentially serve as a novel moonlighting protein capable of performing multiple functions during host-pathogen interactions.

scholarly journals An Autotransporter Protein fromOrientia tsutsugamushiMediates Adherence to Nonphagocytic Host Cells

2011 ◽  
Vol 79 (4) ◽  
pp. 1718-1727 ◽  
Author(s):  
Na-Young Ha ◽  
Nam-Hyuk Cho ◽  
Yeon-Sook Kim ◽  
Myung-Sik Choi ◽  
Ik-Sang Kim
Keyword(s):  
Mammalian Cells ◽  
Scrub Typhus ◽  
Expression System ◽  
Critical Role ◽  
Cellular Adhesion ◽  
Host Cells ◽  
Mammalian Host ◽  
Functional Conservation ◽  
Pulldown Assay ◽  
Obligate Intracellular Pathogen

ABSTRACTOrientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen whose mechanism of cellular adhesion and invasion is poorly characterized. Bioinformatic analyses of twoO. tsutsugamushigenomes revealed the presence of a group of genes that encode autotransporter proteins. In this study, we identified 10 autotransporter gene products and categorized them into five groups of orthologs (ScaA to ScaE) based on their sequence similarities. Sequence homology was highest between members of ScaC group, suggesting the functional conservation of bacterium-host interactions. ScaC was actively expressed on the surface ofO. tsutsugamushiand induced antibody responses in scrub typhus patients. Experiments using microbeads conjugated to recombinant ScaC or a surrogateEscherichia coliexpression system showed that ScaC was sufficient to mediate attachment to, but not invasion of, nonphagocytic mammalian cells. In addition, preincubation of host cells with recombinant ScaC significantly inhibited their interaction withO. tsutsugamushi. Finally, fibronectin was identified as a potential receptor for ScaC by using yeast two-hybrid screening, and this was confirmed using a glutathioneS-transferase (GST) pulldown assay. Taken together, these results demonstrate that ScaC is involved in the interaction ofO. tsutsugamushiwith mammalian host cells and suggest that ScaC may play a critical role in bacterial pathogenesis.

scholarly journals Anaplasma phagocytophilum p44 mRNA Expression Is Differentially Regulated in Mammalian and Tick Host Cells: Involvement of the DNA Binding Protein ApxR

2007 ◽  
Vol 189 (23) ◽  
pp. 8651-8659 ◽  
Author(s):  
Xueqi Wang ◽  
Zhihui Cheng ◽  
Chunbin Zhang ◽  
Takane Kikuchi ◽  
Yasuko Rikihisa
Keyword(s):  
Dna Binding ◽  
Anaplasma Phagocytophilum ◽  
Binding Protein ◽  
Critical Role ◽  
Dna Binding Protein ◽  
Surface Proteins ◽  
Host Cells ◽  
Mammalian Host ◽  
Promoter Regions ◽  
Cells Cultured

ABSTRACT The natural life cycle of Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, consists of alternate infection of two distinct hosts, ticks and mammals, in which bacterial surface proteins are expected to have a critical role. The present study investigated regulation of A. phagocytophilum p44 genes, which encode the P44 major surface proteins. Quantitative real-time reverse transcription-PCR analysis revealed that the amount of p44 mRNA obtained from spleens of A. phagocytophilum-infected SCID mice was approximately 10-fold greater than the amount obtained from salivary glands of A. phagocytophilum-infected Ixodes scapularis nymphs. Similarly, the amount of p44 mRNA obtained from A. phagocytophilum-infected HL-60 cells per bacterium was significantly greater than the amount obtained from infected ISE6 tick cells. The relative amount of p44 mRNA was approximately threefold higher in A. phagocytophilum-infected HL-60 cells cultured at 37°C than in A. phagocytophilum-infected HL-60 cells cultured at 28°C. Although there are more than 100 p44 paralogs, we observed expression mainly from the p44 expression locus (p44E) in various host environments. Interestingly, transcription of the A. phagocytophilum gene encoding the DNA binding protein ApxR was also significantly greater in A. phagocytophilum-infected HL-60 cells than in infected ISE6 tick cells. Gel mobility shift and DNase I protection assays revealed recombinant ApxR binding to the promoter regions of p44E and apxR. ApxR also transactivated the p44E and apxR promoter regions in a lacZ reporter assay. These results indicate that p44 genes and apxR are specifically up-regulated in the mammalian host environment and suggest that ApxR not only is positively autoregulated but also acts as a transcriptional regulator of p44E.

scholarly journals Significant Growth by Rickettsia Species within Human Macrophage-Like Cells Is a Phenotype Correlated with the Ability to Cause Disease in Mammals

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 228
Author(s):  
M. Nathan Kristof ◽  
Paige E. Allen ◽  
Lane D. Yutzy ◽  
Brandon Thibodaux ◽  
Christopher D. Paddock ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Spotted Fever ◽  
Growth Characteristic ◽  
Human Macrophage ◽  
Rickettsia Conorii ◽  
Spotted Fever Group ◽  
Rocky Mountain Spotted Fever ◽  
Phagocytic Cells ◽  
Pathogenic Potential ◽  
Rickettsia Species

Rickettsia are significant sources of tick-borne diseases in humans worldwide. In North America, two species in the spotted fever group of Rickettsia have been conclusively associated with disease of humans: Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, and Rickettsia parkeri, the cause of R. parkeri rickettsiosis. Previous work in our lab demonstrated non-endothelial parasitism by another pathogenic SFG Rickettsia species, Rickettsia conorii, within THP-1-derived macrophages, and we have hypothesized that this growth characteristic may be an underappreciated aspect of rickettsial pathogenesis in mammalian hosts. In this work, we demonstrated that multiple other recognized human pathogenic species of Rickettsia, including R. rickettsii, R. parkeri, Rickettsia africae, and Rickettsiaakari can grow within target endothelial cells as well as within PMA-differentiated THP-1 cells. In contrast, Rickettsia bellii, a Rickettsia species not associated with disease of humans, and R. rickettsii strain Iowa, an avirulent derivative of pathogenic R. rickettsii, could invade both cell types but proliferate only within endothelial cells. Further analysis revealed that similar to previous studies on R. conorii, other recognized pathogenic Rickettsia species could grow within the cytosol of THP-1-derived macrophages and avoided localization with two different markers of lysosomal compartments; LAMP-2 and cathepsin D. R. bellii, on the other hand, demonstrated significant co-localization with lysosomal compartments. Collectively, these findings suggest that the ability of pathogenic rickettsial species to establish a niche within macrophage-like cells could be an important factor in their ability to cause disease in mammals. These findings also suggest that analysis of growth within mammalian phagocytic cells may be useful to predict the pathogenic potential of newly isolated and identified Rickettsia species.

scholarly journals Heterologously Expressed Staphylococcus aureusFibronectin-Binding Proteins Are Sufficient for Invasion of Host Cells

2000 ◽  
Vol 68 (12) ◽  
pp. 6871-6878 ◽  
Author(s):  
Bhanu Sinha ◽  
Patrice Francois ◽  
Yok-Ai Que ◽  
Muzaffar Hussain ◽  
Christine Heilmann ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Binding Proteins ◽  
Surface Protein ◽  
Latex Agglutination ◽  
Surface Proteins ◽  
Host Cells ◽  
Gram Positive ◽  
Fibronectin Receptor ◽  
293 Cells ◽  
Accessible Surface

ABSTRACT Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureusfibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin α5β1 (B. Sinha et al., Cell. Microbiol. 1:101–117, 1999). However, it is unknown whether this mechanism is sufficient for S. aureus invasion. To address this question, various S. aureus adhesins (FnBPA, FnBPB, and clumping factor [ClfA]) were expressed in Staphylococcus carnosus and Lactococcus lactis subsp.cremoris. Both noninvasive gram-positive microorganisms are genetically distinct from S. aureus, lack any knownS. aureus surface protein, and do not bind fibronectin. Transformants of S. carnosus and L. lactisharboring plasmids coding for various S. aureus surface proteins (FnBPA, FnBPB, and ClfA) functionally expressed adhesins (as determined by bacterial clumping in plasma, specific latex agglutination, Western ligand blotting, and binding to immobilized and soluble fibronectin). FnBPA or FnBPB but not of ClfA conferred invasiveness to S. carnosus and L. lactis. Invasion of 293 cells by transformants was comparable to that of strongly invasive S. aureus strain Cowan 1. Binding of soluble and immobilized fibronectin paralleled invasiveness, demonstrating that the amount of accessible surface FnBPs is rate limiting. Thus, S. aureus FnBPs confer invasiveness to noninvasive, apathogenic gram-positive cocci. Furthermore, FnBP-coated polystyrene beads were internalized by 293 cells, demonstrating that FnBPs are sufficient for invasion of host cells without the need for (S. aureus-specific) coreceptors.

scholarly journals Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms

2005 ◽  
Vol 77 (1) ◽  
pp. 77-94 ◽  
Author(s):  
Renato A. Mortara ◽  
Walter K. Andreoli ◽  
Noemi N. Taniwaki ◽  
Adriana B. Fernandes ◽  
Claudio V. da Silva ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Mammalian Cells ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Mammalian Host ◽  
Target Cells ◽  
Sylvatic Cycle ◽  
Final Destination ◽  
Different Strains

Trypanosoma cruzi, the etiological agent of Chagas’ disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.

scholarly journals A streamlined method for transposon mutagenesis ofRickettsia parkeriyields numerous mutations that impact infection

2018 ◽  
Author(s):  
Rebecca L. Lamason ◽  
Natasha M. Kafai ◽  
Matthew D. Welch
Keyword(s):  
Host Cell ◽  
Virulence Factors ◽  
Vascular Endothelial Cells ◽  
Spotted Fever ◽  
Transposon Mutagenesis ◽  
Host Cells ◽  
Spotted Fever Group ◽  
Loss Of Function ◽  
Rickettsia Parkeri ◽  
Obligate Intracellular

AbstractThe rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria,Rickettsiaare highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improvedmariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group speciesRickettsia parkeri. These mutants targeted genes implicated in a variety of pathways, including bacterial replication and metabolism, hypothetical proteins, the type IV secretion system, as well as factors with previously established roles in host cell interactions and pathogenesis. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis.

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