Host Cell Adhesion Research Articles

Curli Proteins CsgE and CsgF Inhibit the Aggregation of Human Islet Amyloid Polypeptide

Curli, a type of proteinaceous cell surface filament, found on enteric bacteria such as E. Coli and Salmonella, are thought to play an important role in host cell adhesion and invasion. The assembly of CsgA, the major structural protein of Curli, into fibers is aided by three non‐structural proteins, CsgE, CsgF, and CsgG. CsgE and CsgF are thought to be periplasmic chaperone proteins that play a vital role in the assembly of Curli fibers. It has been shown that CsgE is able to prevent the aggregation of CsgA into Curli fibers. We determined the ability of CsgE and CsgF to inhibit the aggregation of human Islet Amyloid Polypeptide (hIAPP), an amyloidogenic protein unrelated to Curli formation. We monitored the aggregation of hIAPP, using the fluorescent dye ThT, in the absence and presence of CsgE and CsgF. An increase in ThT fluorescence emission intensity is routinely used to detect the aggregation of amyloidogenic polypeptides. In the absence of CsgE or CsgF we observe a greater than 5‐fold increase in fluorescence intensity when freshly dissolved hIAPP is monitored in the presence of ThT. Such an increase in intensity is typically taken to indicate the aggregation of hIAPP. In the presence of CsgE and CsgF no significant increase in ThT intensity is observed suggesting that both CsgE and CsgF prevented the aggregation of hIAPP.Support or Funding Information1. This work is supported by NIH grant 1R15GM123430‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural and Functional Effects of Altering the Nonpolar Core of Hemolysin A

The protein structure and function paradigm, a foundational tenet of biomolecular science, underlies many infectious diseases. Hemolysin A (HpmA), a hemolytic protein produced by Proteus mirabilis, was used as a model to investigate the protein structure‐function paradigm. HpmA is a member of the two‐partner secretion (TPS) pathway, which is used by gram‐negative bacteria to export predominantly virulent proteins outside of the cell. Through this mechanism, the A‐component (HpmA) is translocated, folded, and activated by its cognate B‐component in the absence of high‐energy bond and electrochemical gradient dependency. All TpsA components are relatively large and can be further divided into two domains, the two‐partner secretion domain and the functional domain. Universally, all known TPS domains harbor a right‐handed, parallel β‐helix architecture and are essential for cognate TpsB‐dependent recognition and secretion. Conversely, functional domains provide TpsA diversification including, cytolysis, host cell adhesion, contact‐growth inhibition, and iron sequestration. A truncated version of hemolysin A (HpmA265) was implemented to define the contributions of the TPS domain toward TpsA structure and function. Recently, our group has further dissected HpmA265 into three sequentially folded structural units termed the polar core, non‐polar core, and carboxy‐terminal subdomains. This research project aims to expand upon our recent results and structurally map the role of the nonpolar core during TPS domain dependent secretion, folding and function. Specifically, residues within the non‐polar core subdomain have been selectively targeted and modified. The structural and functional effects of these site‐selective modifications have been evaluated via chemical denaturation, protease sensitivity and hemolytic assays. Each site‐selective alteration selectively shifts the unfolding transitions attributed to the non‐polar core and carboxy‐terminal subdomain, while leaving the polar core transition unaffected. Furthermore, non‐polar core subdomain destabilization differentially alters secretion levels, while leaving hemolytic activity unaffected. More broadly, these results further define β‐helix TPS domain contribution during gram‐negative bacterial infections like whooping cough, meningitis, and urinary tract infections.Support or Funding InformationFunding for this research was provided by: University Wisconsin – La Crosse Faculty Research Grant Program (TMW) and University Wisconsin – La Crosse Undergraduate Research and Creativity Grant Program (GMS).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AIG1 affects in vitro and in vivo virulence in clinical isolates of Entamoeba histolytica.

The disease state of amebiasis, caused by Entamoeba histolytica, varies from asymptomatic to severe manifestations that include dysentery and extraintestinal abscesses. The virulence factors of the pathogen, and host defense mechanisms, contribute to the outcomes of infection; however, the underlying genetic factors, which affect clinical outcomes, remain to be fully elucidated. To identify these genetic factors in E. histolytica, we used Illumina next-generation sequencing to conduct a comparative genomic analysis of two clinical isolates obtained from diarrheal and asymptomatic patients (strains KU50 and KU27, respectively). By mapping KU50 and KU27 reads to the genome of a reference HM-1:IMSS strain, we identified two genes (EHI_089440 and EHI_176590) that were absent in strain KU27. In KU27, a single AIG1 (avrRpt2-induced gene 1) family gene (EHI_176590) was found to be deleted, from a tandem array of three AIG1 genes, by homologous recombination between the two flanking genes. Overexpression of the EHI_176590 gene, in strain HM-1:IMSS cl6, resulted in increased formation of cell-surface protrusions and enhanced adhesion to human erythrocytes. The EHI_176590 gene was detected by PCR in 56% of stool samples from symptomatic patients infected with E. histolytica, but only in 15% of stool samples from asymptomatic individuals. This suggests that the presence of the EHI_176590 gene is correlated with the outcomes of infection. Taken together, these data strongly indicate that the AIG1 family protein plays a pivotal role in E. histolytica virulence via regulation of host cell adhesion. Our in-vivo experiments, using a hamster liver abscess model, showed that overexpression or gene silencing of EHI_176590 reduced and increased liver abscess formation, respectively. This suggests that the AIG1 genes may have contrasting roles in virulence depending on the genetic background of the parasite and host environment.

Single Molecule Force Spectroscopy Reveals Two-Domain Binding Mode of Pilus-1 Tip Protein RrgA of Streptococcus pneumoniae to Fibronectin.

For host cell adhesion and invasion, surface piliation procures benefits for bacteria. A detailed investigation of how pili adhere to host cells is therefore a key aspect in understanding their role during infection. Streptococcus pneumoniae TIGR 4, a clinical relevant serotype 4 strain, is capable of expressing pilus-1 with terminal RrgA, an adhesin interacting with host extracellular matrix (ECM) proteins. We used single molecule force spectroscopy to investigate the binding of full-length RrgA and single RrgA domains to fibronectin. Our results show that full-length RrgA and its terminal domains D3 and D4 bind to fibronectin with forces of 51.6 (full length), 52.8 (D3), and 46.2 pN (D4) at force-loading rates of around 1500 pN/s. Selective saturation of D3 and D4 binding sites on fibronectin showed that both domains can interact simultaneously with fibronectin, revealing a two-domain binding mechanism for the pilus-1 tip protein. The high off rates and the corresponding short lifetime of the RrgA Fn bond (τ = 0.26 s) may enable piliated pneumococci to form and maintain a transient contact to fibronectin-containing host surfaces and thus to efficiently scan the surface for specific receptors promoting host cell adhesion and invasion. These molecular properties could be essential for S. pneumoniae pili to mediate initial contact to the host cells and-shared with other piliated Gram-positive bacteria-favor host invasion.

A chemical and biological toolbox for Type Vd secretion: Characterization of the phospholipase A1 autotransporter FplA from Fusobacterium nucleatum

Fusobacterium nucleatum is an oral pathogen that is linked to multiple human infections and colorectal cancer. Strikingly, F. nucleatum achieves virulence in the absence of large, multiprotein secretion systems (Types I, II, III, IV, and VI), which are widely used by Gram-negative bacteria for pathogenesis. By contrast, F. nucleatum strains contain genomic expansions of Type V secreted effectors (autotransporters) that are critical for host cell adherence, invasion, and biofilm formation. Here, we present the first characterization of an F. nucleatum Type Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby rename Fusobacterium phospholipase autotransporter (FplA). Biochemical analysis of multiple Fusobacterium strains revealed that FplA is expressed as a full-length 85-kDa outer membrane–embedded protein or as a truncated phospholipase domain that remains associated with the outer membrane. Whereas the role of Type Vd secretion in bacteria remains unidentified, we show that FplA binds with high affinity to host phosphoinositide-signaling lipids, revealing a potential role for this enzyme in establishing an F. nucleatum intracellular niche. To further analyze the role of FplA, we developed an fplA gene knock-out strain, which will guide future in vivo studies to determine its potential role in F. nucleatum pathogenesis. In summary, using recombinant FplA constructs, we have identified a biochemical toolbox that includes lipid substrates for enzymatic assays, potent inhibitors, and chemical probes to detect, track, and characterize the role of Type Vd secreted phospholipases in Gram-negative bacteria.

The Role of Universal Stress Proteins in Bacterial Infections.

Universal stress proteins are ubiquitously expressed in bacteria, archaea and plants and other eukaryotes. A general property of USPs is their role in adaptation of bacteria to oxidative stress, high temperature, low pH and/or hypoxia. There is increasing evidence that these proteins facilitate the adaption of bacterial pathogens to the human host environment, thereby facilitating colonisation and pathogenicity. USPs in Mycobacterium tuberculosis are well studied and may play a role in latency of tuberculosis. USP expressed by Acinetobacter baumannii, Listeria monocytogenes and Salmonella enterica serovar Typhimurium are involved in survival in vivo, while USPs expressed in Pseudomonas aeruginosa and Porphyromonas gingivalis are involved in biofilm formation. Burkholderia cepacia complex and Staphylococcus aureus express USPs that play roles in host cell or host protein adhesion. There is also increasing evidence that USPs also bind to antimicrobial agents and may be ideal candidates to target in the future design of new anti-virulence strategies.

Prevalence of curli genes among Cronobacter species and their roles in biofilm formation and cell-cell aggregation

Cronobacter species are food-borne opportunistic pathogens that cause sepsis, meningitis, and necrotizing enterocolitis in neonates. Bacterial pathogens such as pathogenic Escherichia coli and Salmonella species express extracellular curli fimbriae that are involved in rugosity, biofilm formation, and host cell adherence. csgBAC operon encodes the major curli structural subunit CsgA and the nucleator protein CsgB. csgDEFG operon encodes the regulatory protein CsgD and putative assembly factors. In this study, the curli operons were analyzed in the sequences of 13 Cronobacter strains and other enteric bacterial pathogens. Isogenic mutants of csgA and csgB were generated in C. turicensis LMG23827 (z3032). csgA and csgB mutants did not express curli fimbriae as indicated by a lack of Congo red binding and absence of curli by electron microscopic evaluation. Compared to the wild type strain, biofilm formation and cell-cell aggregation of csgA and csgB mutants were remarkably decreased. The prevalence of curli operons were investigated in 231 Cronobacter strains isolated from different sources using polymerase chain reaction (PCR) assay. The results of the PCR analysis showed that csgA and csgG were present in 30% clinical isolates, 8% food, and 11% environmental isolates. These genes were present in C. dublinensis, C. malonaticus, C. turicensis, and C. universalis, but not in C. muytjensii and C. sakazakii. Our data indicate that csgBAC and csgDEFG operons were present about three fold higher in clinical isolates than in isolates from other sources. The csgA and csgB genes were shown to be involved in the early stages of biofilm development and cell-cell aggregation in Cronobacter.

Genetic evidence for a novel variant of the pilus island 1 backbone protein in group B Streptococcus.

Pili contribute significantly to the pathogenesis of infection of group B Streptococcus (GBS) by facilitating adhesion and invasion of host cells. GBS pilin subunits (the backbone pilin protein, BP, and the ancillary pilin proteins, AP) as well as the specific enzymes required for pilus assembly are encoded by genes located in two separate genomic regions, known as pilus island 1 (PI-1) and PI-2. Our aim was to characterize the pilus profile of a collection of GBS isolates from metropolitan Toronto, Canada. The pilus profile of 1332 invasive and colonizing GBS isolates was determined by PCR and, in selected cases, by whole genome sequencing. While investigating the pilus profile of a collection of GBS organisms, we discovered that 51 isolates possessed a novel variant of the PI-1 BP, which we named BP-1b. The predicted translated sequences of archetypical GBS BP-1 and novel BP-1b variants shared only 63 % amino acid sequence homology. The novel BP-1b variant was most common among strains of serotype Ib and VI, but was also found among strains of serotypes Ia, II, III and VIII. We describe a relatively frequent occurrence of a novel PI-1 BP that cannot be detected by a commonly used multiplex PCR scheme, which could lead to strains being mistyped as PI-1 negative. We present PCR primers that can easily be incorporated into the multiplex PCR assay to identify strains with novel BP-1b variant.

Heterologous expression of Plasmodium vivax apical membrane antigen 1 (PvAMA1) for binding peptide selection.

BackgroundPlasmodium is an obligate intracellular parasite. Apical membrane antigen 1 (AMA1) is the most prominent and well characterized malarial surface antigen that is essential for parasite-host cell invasion, i.e., for sporozoite to invade and replicate within hepatocytes in the liver stage and merozoite to penetrate and replicate within erythrocytes in the blood stage. AMA1 has long served as a potent antimalarial drug target and is a pivotal vaccine candidate. A good understanding of the structure and molecular function of this Plasmodium protein, particularly its involvement in host-cell adhesion and invasion, is of great interest and hence it offers an attractive target for the development of novel therapeutics. The present study aims to heterologous express recombinant Plasmodium AMA1 ectodomain of P. vivax (rPvAMA1) for the selection of binding peptides.MethodsThe rPvAMA1 protein was heterologous expressed using a tag-free Profinity eXactTM system and codon optimized BL21-Codon Plus (DE3)-RIL Escherichia coli strain and further refolded by dialysis for renaturation. Binding peptides toward refolded rPvAMA1 were panned using a Ph.D.-12 random phage display library.ResultsThe rPvAMA1 was successfully expressed and refolded with three phage-displayed dodecapeptides designated as PdV1 (DLTFTVNPLSKA), PdV2 (WHWSWWNPNQLT), and PdV3 (TSVSYINNRHNL) with affinity towards rPvAMA1 identified. All of them exhibited positive binding signal to rPvAMA1 in both direct phage assays, i.e., phage ELISA binding assay and Western blot binding assay.DiscussionPhage display technology enables the mapping of protein-protein interactions based on a simple principle that a library of phage particles displaying peptides is used and the phage clones that bind to the target protein are selected and identified. The binding sites of each selected peptides toward PvAMA1 (Protein Data Bank, PDB ID: 1W8K) were in silico predicted using CABS-dock web server. In this case, the binding peptides provide a valuable starting point for the development of peptidomimetic as antimalarial antagonists directed at PvAMA1.

Fibroblast growth factor receptor-1 mediates internalization of pathogenic spotted fever rickettsiae into host endothelium.

Rickettsial infections continue to cause serious morbidity and mortality in severe human cases around the world. Host cell adhesion and invasion is an essential requisite for intracellular growth, replication, and subsequent dissemination of pathogenic rickettsiae. Heparan sulfate proteoglycans [HSPGs] facilitate the interactions between fibroblast growth factor(s) and their tyrosine kinase receptors resulting in receptor dimerization/activation and have been implicated in bacterial adhesion to target host cells. In the present study, we have investigated the contributions of fibroblast growth factor receptors [FGFRs] in rickettsial entry into the host cells. Inhibition of HSPGs by heparinase and FGFRs by AZD4547 (a selective small-molecule inhibitor) results in significant reduction in rickettsial internalization into cultured human microvascular endothelial cells (ECs), which represent the primary targets of pathogenic rickettsiae during human infections. Administration of AZD4547 during R. conorii infection in a murine model of endothelial-target spotted fever rickettsiosis also diminishes pulmonary rickettsial burden in comparison to mock-treated controls. Silencing of FGFR1 expression using a small interfering RNA also leads to similar inhibition of R. rickettsii invasion into ECs. Consistent with these findings, R. rickettsii infection of ECs also results in phosphorylation of tyrosine 653/654, suggesting activation of FGFR1. Using isobaric tag for relative and absolute quantitation [iTRAQ]-based proteomics approach, we further demonstrate association of β-peptide of rickettsial outer membrane protein OmpA with FGFR1. Mechanistically, FGFR1 binds to caveolin-1 and mediates bacterial entry via caveolin-1 dependent endocytosis. Together, these results identify host cell FGFR1 and rickettsial OmpA as another novel receptor-ligand pair contributing to the internalization of pathogenic rickettsiae into host endothelial cells and the potential application of FGFR-inhibitor drugs as adjunct therapeutics against spotted fever rickettsioses.

Alginate microbeads are coagulation compatible, while alginate microcapsules activate coagulation secondary to complement or directly through FXII.

Alginate microcapsules are prospective candidate materials for cell encapsulation therapy. The material surface must be free of host cell adhesion to ensure free diffusion of nutrition and oxygen to the encapsulated cells. Coagulation activation is one gateway to cellular overgrowth through deposition of fibrin. Herein we used a physiologically relevant whole blood model to investigate the coagulation potential of alginate microcapsules and microbeads. The coagulation potentials and the pathways of activation were depending on the surface properties of the materials. Activation of the complement system could also be involved, thus emphasizing a complement-coagulation cross-talk. Our findings points to complement and coagulation inhibition as intervention point for preventing host reactions, and enhance functional cell-encapsulation devices.

Determination of regional relationships among Salmonella spp. isolated from retail pork circulating in the Chiang Mai municipality area using a WGS data approach

Salmonella is recognized as a significant zoonotic foodborne pathogen, and pork products are involved in one-fifth of infections. Whole genome sequencing data of Salmonella isolated from retail's pork circulating in the Chiang Mai Municipality area between April 2013 and September 2014, were used to focus on genetic diversity and proven in pig-human transmission based on Multilocus Sequence Typing (MLST). Additionally, WGS data were used to investigate virulence genes, to assess the hazard or pathogenic potential transferred into the food production chain. In this study, all 32 Salmonella strains were classified into 11 Sequence Types (STs). ST469 accounted for the majority (41%). The sequence types of two other strains, 6% of the total, could not be identified. All tested strains carried at least 15 virulence genes. The most frequent gene profile was “sfm-fim-sop-inv.-org-sip-spa-sif-fli-flg-hil-spr-ssa-sse-pag-bss” (47%). Salmonella circulating in the study area demonstrated competence in biofilm production, host cell adhesion, host cell invasion, and host cell survival. Based on the phenotypic and genotypic findings, as well as pathogen source, it appears possible that a common supply chain or common infection source might be presented in the retail pork system in the study area. In addition, an epidemiological comparison of the Salmonella genotypes from the current study with those from other areas such as People's Republic of China (PR China) and the Lao People's Democratic Republic (Lao PDR) was generated by Minimum spanning tree (MST). Identical strains originating from humans, animals and food were found. The findings indicate that contamination can be occured at all levels including pre-harvest, the farm-slaughterhouse-retail chain and consumers over different geographical areas. Acquiring information about infection sources and transmission routes will hopefully motivate all sectors to enforce strict sanitation controls at all production stages including the consumer level.

The Plasmodium rhoptry associated protein complex is important for parasitophorous vacuole membrane structure and intraerythrocytic parasite growth.

Plasmodium parasites must invade erythrocytes in order to cause the disease malaria. The invasion process involves the coordinated secretion of parasite proteins from apical organelles that include the rhoptries. The rhoptry is comprised of two compartments: the neck and the bulb. Rhoptry neck proteins are involved in host cell adhesion and formation of the tight junction that forms between the invading parasite and erythrocyte, whereas the role of rhoptry bulb proteins remains ill-defined due to the lack of functional studies. In this study, we show that the rhoptry-associated protein (RAP) complex is not required for rhoptry morphology or erythrocyte invasion. Instead, post-invasion when the parasite is bounded by a parasitophorous vacuolar membrane (PVM), the RAP complex facilitates the survival of the parasite in its new intracellular environment. Consequently, conditional knockdown of members of the RAP complex leads to altered PVM structure, delayed intra-erythrocytic growth, and reduced parasitaemias in infected mice. This study provides evidence that rhoptry bulb proteins localising to the parasite-host cell interface are not simply by-products of the invasion process but contribute to the growth of Plasmodium in vivo.

Role of RNase Y in Clostridium perfringens mRNA Decay and Processing.

RNases have important roles in RNA degradation and turnover in all organisms. C. perfringens is a Gram-positive anaerobic spore-forming bacterial pathogen that produces numerous extracellular enzymes and toxins, and it is linked to digestive disorders and disease. A highly conserved endoribonuclease, RNase Y, affects the expression of hundreds of genes, including toxin genes, and studying these effects is useful for understanding C. perfringens specifically and RNases generally. Moreover, RNase Y is involved in processing specific transcripts, and we observed that this processing in C. perfringens results in the stabilization of mRNAs encoding a toxin and bacterial extracellular apparatus pili. Our study shows that RNase activity is associated with gene expression, helping to determine the growth, proliferation, and virulence of C. perfringens.

Pyruvate dehydrogenase subunit β of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation.

Multi-functional surface proteins have been observed in a variety of pathogenic bacteria, where they mediate host cell adhesion and invasion, as well as in commensal bacterial species, were they mediate positive interaction with the host. Among these proteins, some glycolytic enzymes, expressed on the bacterial cell surface, can bind human extracellular matrix components (ECM). A major target for them is collagen, an abundant glycoprotein of connective tissues. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is involved in binding with collagen type I (CnI). In this study, we found that PDHB, a component of the pyruvate dehydrogenase complex, contributes to the L. plantarum LM3 adhesion to CnI. By a cellular adhesion assay to immobilized CnI, we show that LM3-B1 cells, carrying a null mutation in the pdhB gene, bind to CnI - coated surfaces less efficiently than wild-type cells. Moreover, we show that the PDHB-CnI interaction requires a native state for PDHB. We also analyzed the ability to develop biofilm in wild-type and mutant strains and we found that the lack of the PDHB on cell surface generates cells partially impaired in biofilm development.

The Group A Streptococcus serotype M2 pilus plays a role in host cell adhesion and immune evasion.

Group A Streptococcus (GAS), or Streptococcus pyogenes, is a human pathogen that causes diseases ranging from skin and soft tissue infections to severe invasive diseases, such as toxic shock syndrome. Each GAS strain carries a particular pilus type encoded in the variable fibronectin-binding, collagen-binding, T antigen (FCT) genomic region. Here, we describe the functional analysis of the serotype M2 pilus encoded in the FCT-6 region. We found that, in contrast to other investigated GAS pili, the ancillary pilin 1 lacks adhesive properties. Instead, the backbone pilin is important for host cell adhesion and binds several host factors, including fibronectin and fibrinogen. Using a panel of recombinant pilus proteins, GAS gene deletion mutants and Lactococcus lactis gain-of-function mutants we show that, unlike other GAS pili, the FCT-6 pilus also contributes to immune evasion. This was demonstrated by a delay in blood clotting, increased intracellular survival of the bacteria in macrophages, higher bacterial survival rates in human whole blood and greater virulence in a Galleria mellonella infection model in the presence of fully assembled FCT-6 pili.

The expression of NTPDase1 and -2 of Leishmania infantum chagasi in bacterial and mammalian cells: Comparative expression, refolding and nucleotidase characterization

Visceral Leishmaniasis (VL) represents an important global health problem in several warm countries around the world. The main targets in this study are the two nucleoside triphosphate diphosphohydrolases (NTPDases) from Leishmania infantum chagasi that are the main etiologic agent of VL in the New World. These enzymes, called LicNTPDase1 and -2, are homologous to members 5 and 6 of the mammalian E-NTPDase/CD39 superfamily of enzymes. These enzymes hydrolyze nucleotides and accordingly can participate in the purine salvage pathways and in the modulation of purinergic signaling through the extracellular nucleotide-dependent host immune responses. They can therefore affect adhesion and infection of host cells and the parasite virulence. To further characterize these enzymes, in this work, we expressed LicNTPDase1 and -2 in the classical bacterial system Escherichia coli and mammalian cell system COS-7 cells. Our data demonstrate that changes in refolding after expression in bacteria can increase the activity of recombinant (r) rLicNTPDase2 up to 20 times but has no significant effect on rLicNTPDase1. Meanwhile, the expression in COS-7 led to a significant increase in activity for rLicNTPDase1.

Structural Diversity in the Type IV Pili of Multidrug-resistant Acinetobacter

Acinetobacter baumannii is a Gram-negative coccobacillus found primarily in hospital settings that has recently emerged as a source of hospital-acquired infections. A. baumannii expresses a variety of virulence factors, including type IV pili, bacterial extracellular appendages often essential for attachment to host cells. Here, we report the high resolution structures of the major pilin subunit, PilA, from three Acinetobacter strains, demonstrating that A. baumannii subsets produce morphologically distinct type IV pilin glycoproteins. We examine the consequences of this heterogeneity for protein folding and assembly as well as host-cell adhesion by Acinetobacter Comparisons of genomic and structural data with pilin proteins from other species of soil gammaproteobacteria suggest that these structural differences stem from evolutionary pressure that has resulted in three distinct classes of type IVa pilins, each found in multiple species.

Structural Insights into Substrate Recognition and Catalysis in Outer Membrane Protein B (OmpB) by Protein-lysine Methyltransferases from Rickettsia

Rickettsia belong to a family of Gram-negative obligate intracellular infectious bacteria that are the causative agents of typhus and spotted fever. Outer membrane protein B (OmpB) occurs in all rickettsial species, serves as a protective envelope, mediates host cell adhesion and invasion, and is a major immunodominant antigen. OmpBs from virulent strains contain multiple trimethylated lysine residues, whereas the avirulent strain contains mainly monomethyllysine. Two protein-lysine methyltransferases (PKMTs) that catalyze methylation of recombinant OmpB at multiple sites with varying sequences have been identified and overexpressed. PKMT1 catalyzes predominantly monomethylation, whereas PKMT2 catalyzes mainly trimethylation. Rickettsial PKMT1 and PKMT2 are unusual in that their primary substrate appears to be limited to OmpB, and both are capable of methylating multiple lysyl residues with broad sequence specificity. Here we report the crystal structures of PKMT1 from Rickettsia prowazekii and PKMT2 from Rickettsia typhi, both the apo form and in complex with its cofactor S-adenosylmethionine or S-adenosylhomocysteine. The structure of PKMT1 in complex with S-adenosylhomocysteine is solved to a resolution of 1.9 Å. Both enzymes are dimeric with each monomer containing an S-adenosylmethionine binding domain with a core Rossmann fold, a dimerization domain, a middle domain, a C-terminal domain, and a centrally located open cavity. Based on the crystal structures, residues involved in catalysis, cofactor binding, and substrate interactions were examined using site-directed mutagenesis followed by steady state kinetic analysis to ascertain their catalytic functions in solution. Together, our data reveal new structural and mechanistic insights into how rickettsial methyltransferases catalyze OmpB methylation.

Trypanosoma cruzi Invasion into Host Cells: A Complex Molecular Targets Interplay.

Chagas' disease is still a worldwide threat, with estimated from 6 to 7 million infected people, mainly in Latin America. Despite all efforts, especially from international consortia (DNDi, NMTrypI), to develop an innovative therapeutic strategy against this disease, no candidate has achieved full requirements for clinical use yet. In this review, we point out the general molecular and cellular mechanisms involved in T. cruzi cell invasion and elucidate the roles of specific parasite and host targets in the progress of Chagas' disease. Among these molecular targets are Gp85/transsialidase, mucins, cruzipain and oligopeptidase B, found in parasite cell surface, and Galectin-3 and Toll-like receptors present in host cells. Thus, the deep understanding of their interplay and involvement on T. cruzi host cell adhesion, invasion and evasion from host immune may expand the chances for discovering new therapeutic agents against this neglected disease. Additionally, these targets may represent a remarkable strategy to block parasite invasion in the early stages of infection.