Pilus Protein Research Articles

Global Proteomic Profiling of Piscirickettsia salmonis and Salmon Macrophage-Like Cells during Intracellular Infection.

Piscirickettsiasalmonis is an intracellular bacterial fish pathogen that causes piscirickettsiosis, a disease with numerous negative impacts in the Chilean salmon farming industry. Although transcriptomic studies of P. salmonis and its host have been performed, dual host–pathogen proteomic approaches during infection are still missing. Considering that gene expression does not always correspond with observed phenotype, and bacteriological culture studies inadequately reflect infection conditions, to improve the existing knowledge for the pathogenicity of P. salmonis, we present here a global proteomic profiling of Salmon salar macrophage-like cell cultures infected with P. salmonis LF-89. The proteomic analyses identified several P. salmonis proteins from two temporally different stages of macrophages infection, some of them related to key functions for bacterial survival in other intracellular pathogens. Metabolic differences were observed in early-stage infection bacteria, compared to late-stage infections. Virulence factors related to membrane, lipopolysaccharide (LPS) and surface component modifications, cell motility, toxins, and secretion systems also varied between the infection stages. Pilus proteins, beta-hemolysin, and the type VI secretion system (T6SS) were characteristic of the early-infection stage, while fimbria, upregulation of 10 toxins or effector proteins, and the Dot/Icm type IV secretion system (T4SS) were representative of the late-infection stage bacteria. Previously described virulence-related genes in P. salmonis plasmids were identified by proteomic assays during infection in SHK-1 cells, accompanied by an increase of mobile-related elements. By comparing the infected and un-infected proteome of SHK-1 cells, we observed changes in cellular and redox homeostasis; innate immune response; microtubules and actin cytoskeleton organization and dynamics; alteration in phagosome components, iron transport, and metabolism; and amino acids, nucleoside, and nucleotide metabolism, together with an overall energy and ATP production alteration. Our global proteomic profiling and the current knowledge of the P. salmonis infection process allowed us to propose a model of the macrophage–P. salmonis interaction.

Deciphering Streptococcal Biofilms.

Streptococci are a diverse group of bacteria, which are mostly commensals but also cause a considerable proportion of life-threatening infections. They colonize many different host niches such as the oral cavity, the respiratory, gastrointestinal, and urogenital tract. While these host compartments impose different environmental conditions, many streptococci form biofilms on mucosal membranes facilitating their prolonged survival. In response to environmental conditions or stimuli, bacteria experience profound physiologic and metabolic changes during biofilm formation. While investigating bacterial cells under planktonic and biofilm conditions, various genes have been identified that are important for the initial step of biofilm formation. Expression patterns of these genes during the transition from planktonic to biofilm growth suggest a highly regulated and complex process. Biofilms as a bacterial survival strategy allow evasion of host immunity and protection against antibiotic therapy. However, the exact mechanisms by which biofilm-associated bacteria cause disease are poorly understood. Therefore, advanced molecular techniques are employed to identify gene(s) or protein(s) as targets for the development of antibiofilm therapeutic approaches. We review our current understanding of biofilm formation in different streptococci and how biofilm production may alter virulence-associated characteristics of these species. In addition, we have summarized the role of surface proteins especially pili proteins in biofilm formation. This review will provide an overview of strategies which may be exploited for developing novel approaches against biofilm-related streptococcal infections.

Adhesion of Escherichia Coli to Nanostructured Surfaces and the Role of Type 1 Fimbriae.

Bacterial fimbriae are an important virulence factor mediating adhesion to both biotic and abiotic surfaces and facilitating biofilm formation. The expression of type 1 fimbriae of Escherichia coli is a key virulence factor for urinary tract infections and catheter-associated urinary tract infections, which represent the most common nosocomial infections. New strategies to reduce adhesion of bacteria to surfaces is therefore warranted. The aim of the present study was to investigate how surfaces with different nanotopography-influenced fimbriae-mediated adhesion. Surfaces with three different nanopattern surface coverages made in polycarbonate were fabricated by injection molding from electron beam lithography nanopatterned templates. The surfaces were constructed with features of approximately 40 nm width and 25 nm height with 100 nm, 250 nm, and 500 nm interspace distance, respectively. The role of fimbriae type 1-mediated adhesion was investigated using the E. coli wild type BW25113 and ΔfimA (with a knockout of major pilus protein FimA) and ΔfimH (with a knockout of minor protein FimH) mutants. For the surfaces with nanotopography, all strains adhered least to areas with the largest interpillar distance (500 nm). For the E. coli wild type, no difference in adhesion between surfaces without pillars and the largest interpillar distance was observed. For the deletion mutants, increased adhesion was observed for surfaces without pillars compared to surfaces with the largest interpillar distance. The presence of a fully functional type 1 fimbria decreased the bacterial adhesion to the nanopatterned surfaces in comparison to the mutants.

Association of Combined Sero-Positivity to Helicobacter pylori and Streptococcus gallolyticus with Risk of Colorectal Cancer.

Previously, we found that risk of colorectal cancer (CRC) is increased in individuals with serum antibody response to both Helicobacter pylori (HP) Vacuolating Cytotoxin (VacA) toxin or Streptococcus gallolyticus (SGG) pilus protein Gallo2178. In the present analysis, we tested the hypothesis that combined seropositivity to both antigens is a better indicator of CRC risk than seropositivity to single antigens. We used multiplex serologic assays to analyze pre-diagnostic serum for antibody responses from 4063 incident CRC cases and 4063 matched controls from 10 US cohorts. To examine whether combined SGG Gallo2178 and HP VacA sero-status was associated with CRC risk, we used conditional logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Compared to dual sero-negative individuals, there was no increased risk for individuals sero-positive to SGG Gallo2178 only (OR: 0.93; 95% CI: 0.66–1.31) or to HP VacA only (OR: 1.08; 95% CI: 0.98–1.19). However, dual sero-positive individuals had a >50% increased odds of developing CRC (OR: 1.54; 95% CI: 1.16–2.04), suggesting an interaction between antibody responses to these two pathogens and CRC risk (pinteraction = 0.06). In conclusion, this study suggests that dual sero-positivity to HP VacA and SGG Gallo2178 is an indicator of increased risk of CRC.

Protective effects of egg yolk immunoglobulins (IgYs) developed against recombinant immunogens CtxB, OmpW and TcpA on infant mice infected with Vibrio cholerae

Vibrio cholerae causes cholera and other infections, especially in children under five years of age. Cholera toxin (CT), toxin-coregulated pilus (TCP) and outer membrane protein W (OmpW) are three major virulence factors of this bacterium. The emergence of antimicrobial-resistant (AMR) strains and the absence of a comprehensive and flawless vaccine, has prompted other treatments. There are several advantages of egg yolk antibodies (IgY) over other immunotherapy agents, such as economic feasibility, high yield simple production, and better immune responsiveness to mammalian antigens due to phylogenetic distance. Accordingly, in the present study, IgYs against recombinant proteins CtxB (responsible for the CT binding to eukaryotic cells), TcpA (enhances bacterial attachment to enterocytes) and OmpW were produced, in single, coupled or combined forms, to evaluate and compare their protectivity potency. Immunoreactivity of IgYs were examined through protein and whole cell ELISA, their specificity was confirmed by western blotting, and their neutralizing effects on CT was evaluated in Y1 cell culture. Produced IgYs were gavage administered to different groups of infant mice infected with V. cholerae. The results indicated that IgYs produced against CtxB had the highest titers, and were able to neutralize cytotoxicity effects in Y1 cell culture, while the highest protection in the mice challenge was obtained by IgY-TcpA. No considerable increase was observed in immunoreactivity or protectivity of antibodies produced against combined antigens. The produced IgYs showed a good antigen-specificity and protectivity which can be used in passive immunotherapy against cholera.

Four Chromosomal Type IV Secretion Systems in Helicobacter pylori: Composition, Structure and Function.

The pathogenic bacterium Helicobacter pylori is genetically highly diverse and a major risk factor for the development of peptic ulcer disease and gastric adenocarcinoma in humans. During evolution, H. pylori has acquired multiple type IV secretion systems (T4SSs), and then adapted for various purposes. These T4SSs represent remarkable molecular transporter machines, often associated with an extracellular pilus structure present in many bacteria, which are commonly composed of multiple structural proteins spanning the inner and outer membranes. By definition, these T4SSs exhibit central functions mediated through the contact-dependent conjugative transfer of mobile DNA elements, the contact-independent release and uptake of DNA into and from the extracellular environment as well as the secretion of effector proteins in mammalian host target cells. In recent years, numerous features on the molecular functionality of these T4SSs were disclosed. H. pylori encodes up to four T4SSs on its chromosome, namely the Cag T4SS present in the cag pathogenicity island (cagPAI), the ComB system, as well as the Tfs3 and Tfs4 T4SSs, some of which exhibit unique T4SS functions. The Cag T4SS facilitates the delivery of the CagA effector protein and pro-inflammatory signal transduction through translocated ADP-heptose and chromosomal DNA, while various structural pilus proteins can target host cell receptors such as integrins or TLR5. The ComB apparatus mediates the import of free DNA from the extracellular milieu, whereas Tfs3 may accomplish the secretion or translocation of effector protein CtkA. Both Tfs3 and Tfs4 are furthermore presumed to act as conjugative DNA transfer machineries due to the presence of tyrosine recombinases with cognate recognition sequences, conjugational relaxases, and potential origins of transfer (oriT) found within the tfs3 and tfs4 genome islands. In addition, some extrachromosomal plasmids, transposons and phages have been discovered in multiple H. pylori isolates. The genetic exchange mediated by DNA mobilization events of chromosomal genes and plasmids combined with recombination events could account for much of the genetic diversity found in H. pylori. In this review, we highlight our current knowledge on the four T4SSs and the involved mechanisms with consequences for H. pylori adaptation to the hostile environment in the human stomach.

Type I pili, CsuA/B and FimA induce a protective immune response against Acinetobacter baumannii

BackgroundAcinetobacter baumannii, a nosocomial pathogen, is considered as a common cause of hospital and community-acquired infections. Emerging multidrug-resistance in this pathogen followed by subsequent problems in treatment has been increasing to alarming levels that warrant investigation of new therapeutic approaches. One strategy to reduce antibiotic resistance is to use of vaccines. Although there is no vaccine currently in development for this pathogen, different attempts have been made to develop one. MethodsIn this study, we used two different recombinant pilus proteins (CsuA/B and FimA) either singly or in combination to evaluate protective efficacy against A. baumannii in lethal and sub-lethal murine sepsis models. FindingsActive immunization with recombinant proteins in combination elicited high levels of IgG antibody after the first immunization and provided 62% (five of eight mice; p < 0·001) protection against a lethal dose (1·2 × 106 CFU) of A. baumannii along with efficient clearance of bacteria in internal organs viz. spleen, liver, and lungs at sub-lethal challenge. Immunization with CsuA/B alone conferred partial protection as demonstrated by low survival rate (three [37%] of eight mice; p < 0·05) after lethal challenge and reduction of bacteria in internal organs of the mice after 24 h post-sub-lethal infection. Immunization with FimA, in comparison to CsuA/B, showed better protection (four [50%] of eight mice; p < 0·01) and reduction in CFU after 14 h. InterpretationOur results showed that pilus proteins in combination as a single immunogen could potentially impart protection against A. baumannii. FundingShahed University.

Characterization of Staphylococci and Streptococci Isolated from Milk of Bovides with Mastitis in Egypt.

The aim of this study was to characterize staphylococci and streptococci in milk from Egyptian bovides. In total, 50 milk samples were collected from localities in the Nile Delta region of Egypt. Isolates were cultivated, identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistance-associated genes. Thirty-eight Staphylococcus isolates and six Streptococcus isolates could be cultivated. Staphylococcus aureus isolates revealed a high resistance rate to penicillin, ampicillin, clindamycin, and erythromycin. The mecA gene defining methicillin-resistant Staphylococcus aureus, erm(C) and aac-aphD genes was found in 87.5% of each. Coagulase-negative staphylococci showed a high prevalence of mecA, blaZ and tetK genes. Other resistance-associated genes were found. All Streptococcus dysgalactiae isolates carried blaZ, erm(A), erm(B), erm(C) and lnuA genes, while Streptococcus suis harbored erm(C), aphA-3, tetL and tetM genes, additionally. In Streptococcus gallolyticus, most of these genes were found. The Streptococcus agalactiae isolate harbored blaZ, erm(B), erm(C), lnuA, tetK, tetL and tetM genes. Streptococcus agalactiae isolate was analyzed by DNA microarray analysis. It was determined as sequence type 14, belonging to clonal complex 19 and represented capsule type VI. Pilus and cell wall protein genes, pavA, cadD and emrB/qacA genes were identified by microarray analysis.

A practical overview of molecular replacement: Clostridioides difficile PilA1, a difficult case study.

Many biologists are now routinely seeking to determine the three-dimensional structures of their proteins of choice, illustrating the importance of this knowledge, but also of the simplification and streamlining of structure-determination processes. Despite the fact that most software packages offer simple pipelines, for the non-expert navigating the outputs and understanding the key aspects can be daunting. Here, the structure determination of the type IV pili (TFP) protein PilA1 from Clostridioides difficile is used to illustrate the different steps involved, the key decision criteria and important considerations when using the most common pipelines and software. Molecular-replacement pipelines within CCP4i2 are presented to illustrate the more commonly used processes. Previous knowledge of the biology and structure of TFP pilins, particularly the presence of a long, N-terminal α-helix required for pilus formation, allowed informed decisions to be made during the structure-determination strategy. The PilA1 structure was finally successfully determined using ARCIMBOLDO and the ab initio MR strategy used is described.

High Force Magnetic Tweezers Reveal That Bacterial Adhesion Pili Act as Megadalton-scale Schock Absorbers

Gram positive pathogens colonize the oral cavity with their adhesive pili. These organelles are exposed to large mechanical perturbations, such as mastication or teeth brushing, which constantly challenge the bacterial adhesion. To survive these events, the pilus proteins contain intramolecular isopeptide bonds that confer them high mechanical stability. In the dental plaque pathogen Actinomyces oris, two strategically located isopeptide bonds in the 2nd and the 3rd domain of the pilus protein FimA prevent the mechanical unfolding of this protein. The only structure that can be stretched under force is the 40 residue sequence trapped between both isopeptide bonds, the isopeptide-delimited loop (IDL) motif. Our previous AFM force spectroscopy experiments unveiled the large forces (>600 pN) required to stretch FimA's IDL; however, the low force resolution of AFM prevented us from studying the IDL folding at low forces. Herein, we develop a high force magnetic tweezers force spectroscopy assay to expose FimA's IDL to forces ranging from 4 to 280 pN, allowing us to cover the IDL (un)folding dynamics with nm and sub-pN resolution. Our approach permits us to detect the IDL extension in a few seconds at forces >200 pN, and to monitor IDL folding at forces <15 pN. After IDL folding, FimA mechanical stability increases along time, suggesting a time-dependent maturation of the interfacial contacts established between FimA's 2nd and 3rd domains. Stretching-relaxation experiments reveal that a pilus composed of 150 FimA subunits would be able to dissipate as heat up to ∼4·105 zJ, acting as a megaDalton-scale shock-absorber. Given the ubiquitous presence of this protein motif among the pili of Gram positive bacteria, IDL pharmaceutical targeting could become a strategy to weaken the adhesion resistance of these pathogens.

An extract of protein pili and filtrat of human spermatozoa: A chemical review

This study aims to determine the effect of E. coli pili protein, filtrate media culture of E. coli and mixture of E. coli pili protein and E. coli filtrate on the motility and vitality of human sperm in an in vitro. The method for obtaining E. coli pili protein is E. coli prepared by do culture on TCG media to grow E. coli pili, then cutting the Pili E. coli using Omni mixer. Pili that has been obtained then dialysis, electrophoresis and electroelution. E. coli filtrate was obtained by the method of culturing E. coli on Mc Conkey media for 48 hours and then doing culture on BHI media for 48 hours. E. coli culture on BHI media was then centrifuged. E. coli cultures in BHI media were centrifuged and the supernatant produced from centrifugation was filtered using a. 0.22µm milipore filter. 10 samples of normal donor sperm according to 1999 WHO criteria, in preparation using Percoll method. Spermatozoa were incubated with E. coli pili protein, E. coli filtrate and a mixture of E. coli pili proteins and E. coli filtrate on U-base microplate. Incubation was performed for 1 hour. Observations by calculating the motility and vitality of spermatozoa each at 100 spermatozoa under a light microscope. Data analysis was done with SPSS. The results of this study found that Protein Pili E. coli, E. coli filtrate and a mixture of E. coli pili proteins and E. coli filtrate had significant effect on sperm motility (p = 0.000) and on sperm vitality (p = 0.000). Thus it can be concluded that E. coli pili protein and E. coli filtrate are potentially used for spermicidal materials.

Intranasal immunization with 54 kDa hemagglutinin pili protein of Streptococcus pneumonia increases mucosal and systemic antibodies

Intranasal immunization with 54 kDa hemagglutinin pili protein of Streptococcus pneumonia increases mucosal and systemic antibodies

The adhesive PitA pilus protein from the early dental plaque colonizer Streptococcus oralis: expression, purification, crystallization and X-ray diffraction analysis.

PitA is the putative tip adhesin of the pilus islet 2 (PI-2)-encoded sortase-dependent pilus in the Gram-positive Streptococcus oralis, an opportunistic pathogen that often flourishes within the diseased human oral cavity. Early colonization by S. oralis and its interaction with Actinomyces oris seeds the development of oral biofilm or dental plaque. Here, the PI-2 pilus plays a vital role in mediating adherence to host surfaces and other bacteria. A recombinant form of the PitA adhesin has now been produced and crystallized. Owing to the large size (∼100 kDa), flexibility and complicated folding of PitA, obtaining diffraction-quality crystals has been a challenge. However, by the use of limited proteolysis with α-chymotrypsin, the diffraction quality of the PitA crystals was considerably enhanced to 2.16 Å resolution. These crystals belonged to space group P1, with unit-cell parameters a = 61.48, b = 70.87, c=82.46 Å, α = 80.08, β=87.02, γ = 87.70°. The anomalous signal from the terbium derivative of α-chymotrypsin-treated PitA crystals prepared with terbium crystallophore (Tb-Xo4) was sufficient to obtain an interpretable electron-density map via terbium SAD phasing.

Therapeutic Approaches Targeting the Assembly and Function of Chaperone-Usher Pili.

The chaperone-usher (CU) pathway is a conserved secretion system dedicated to the assembly of a superfamily of virulence-associated surface structures by a wide range of Gram-negative bacteria. Pilus biogenesis by the CU pathway requires two specialized assembly components: a dedicated periplasmic chaperone and an integral outer membrane assembly and secretion platform termed the usher. The CU pathway assembles a variety of surface fibers, ranging from thin, flexible filaments to rigid, rod-like organelles. Pili typically act as adhesins and function as virulence factors that mediate contact with host cells and colonization of host tissues. Pilus-mediated adhesion is critical for early stages of infection, allowing bacteria to establish a foothold within the host. Pili are also involved in modulation of host cell signaling pathways, bacterial invasion into host cells, and biofilm formation. Pili are critical for initiating and sustaining infection and thus represent attractive targets for the development of antivirulence therapeutics. Such therapeutics offer a promising alternative to broad-spectrum antibiotics and provide a means to combat antibiotic resistance and treat infection while preserving the beneficial microbiota. A number of strategies have been taken to develop antipilus therapeutics, including vaccines against pilus proteins, competitive inhibitors of pilus-mediated adhesion, and small molecules that disrupt pilus biogenesis. Here we provide an overview of the function and assembly of CU pili and describe current efforts aimed at interfering with these critical virulence structures.

Prising open the human brain

Cell Biology Meningococcus ( Neisseria meningitidis ) causes meningitis and rapidly progressing fatal shock, but only in humans. To invade the brain, meningococcus uses its filamentous pili to hijack the β2-adrenergic receptor (β2AR), inducing an allosteric β-arrestin–biased signaling cascade in endothelial cells lining the capillaries of the brain. This cascade allows bacterial colonies to tether to endothelial cells, despite the shear stress of blood flow, and also promotes opening of endothelial junctions, which allows bacteria to penetrate the brain. Virion et al. sought to understand how a G protein–coupled receptor is activated by bacterial type IV pili proteins to transduce a signaling cascade that normally needs a cognate ligand. They found that β2AR activation requires two asparagine-branched glycan chains with terminally exposed sialic acid residues. Meningococcus triggers receptor signaling by exerting mechanical forces on β2AR glycans with its retractable pili. Because human glycans are unusual in exposing sialic acid residues on their glycans, this mechanism may help explain the specificity of meningococcus to its human host. Nat. Commun. 10 , 4752 (2019).

Nasopharyngeal Colonization with Streptococcus Pneumoniae in Healthy Infants Less Than 60 Days Attending the Outpatient Clinic of Alexandria University Children’s Hospital

<i>Streptococcus pneumoniae</i> (pneumococcus) is a bacterial pathogen that affects children and adults worldwide [1]. Although S. pneumoniae is the leading bacterial cause of respiratory tract infections, it is also capable of causing a wide variety of infectious syndromes including meningitis, peritonitis, and sepsis [2-4]. Virulence factors include capsule, pnemolysin, the Pneumococcal Pilus, Choline-Binding Proteins (CBPs) and pneumococcal surface proteins and other surface proteins [6].

The type IV pilus protein PilU functions as a PilT-dependent retraction ATPase.

Type IV pili are dynamic cell surface appendages found throughout the bacteria. The ability of these structures to undergo repetitive cycles of extension and retraction underpins their crucial roles in adhesion, motility and natural competence for transformation. In the best-studied systems a dedicated retraction ATPase PilT powers pilus retraction. Curiously, a second presumed retraction ATPase PilU is often encoded immediately downstream of pilT. However, despite the presence of two potential retraction ATPases, pilT deletions lead to a total loss of pilus function, raising the question of why PilU fails to take over. Here, using the DNA-uptake pilus and mannose-sensitive haemagglutinin (MSHA) pilus of Vibrio cholerae as model systems, we show that inactivated PilT variants, defective for either ATP-binding or hydrolysis, have unexpected intermediate phenotypes that are PilU-dependent. In addition to demonstrating that PilU can function as a bona fide retraction ATPase, we go on to make the surprising discovery that PilU functions exclusively in a PilT-dependent manner and identify a naturally occurring pandemic V. cholerae PilT variant that renders PilU essential for pilus function. Finally, we show that Pseudomonas aeruginosa PilU also functions as a PilT-dependent retraction ATPase, providing evidence that the functional coupling between PilT and PilU could be a widespread mechanism for optimal pilus retraction.

Antibody responses to flagellin C and Streptococcus gallolyticus pilus proteins in colorectal cancer

Antibodies to Streptococcus gallolyticus subspecies gallolyticus (SGG) have been associated with colorectal cancer (CRC). Because SGG may correlate with impaired gut epithelia, we assessed the association of antibodies to bacterial flagellin C (FliC), a measure potentially related to this impairment, with CRC and the CRC-specific interaction with antibodies to SGG proteins. Antibodies to FliC and SGG pilus proteins Gallo2178 and Gallo2179 were measured in two independent studies, a combined study from Nijmegen and Detroit (93 CRC cases, 74 controls) and a replication data set including 576 cases and 576 controls from the Spanish multicenter multicase-control study (MCC-Spain). Logistic regression was applied to assess whether antibodies to FliC were associated with CRC and modified the association of antibodies to SGG proteins with CRC. Antibodies to FliC were associated with those to SGG Gallo2178 among CRC cases, resulting in an interaction in the association of antibodies to Gallo2178 with CRC (p = 0.007). This association was only present among individuals with high antibody responses to FliC (OR: 2.42, 95% CI: 1.45–4.06). In conclusion, our findings suggest that colorectal tumorigenesis could be accompanied by an impaired integrity of the epithelium that could result in associated increased antibody responses to bacterial proteins.

The Role of Pili Protein 38,6 kDa Klebsiella pneumoniae as a Hemagglutinin and Adhesin Protein which Serves as a Virulence Factor

Klebsiella pneumoniae is an infectious bacteria in various parts of the body. The presence of proteins in pili that will bind to cell surface receptors (adhesin proteins) and cell membrane sugar molecules (hemagglutinin proteins) is a very influential factor in the ability of K. pneumoniae to enter the human body. The purpose of this study was to determine the role of pili 38.6 kDa K. pneumoniae protein as a hemagglutinin and adhesin protein which functions as a virulence factor. This type of research is pure experimental research using the electrophoresis method (SDS-PAGE) to obtain protein. The protein obtained was then tested for hemagglutination and adhesion test using erythrocyte cells and enterocytes of BALB / C mice to determine their role as hemagglutinin and adhesin proteins. Conclusion of this study pili protein 38.6 kDa Klebsiella pneumoniae acts as a hemagglutinin and adhesion protein which functions as a virulence factor.&#x0D; Keywords: Klebsiella pneumonia, 38,6 kDa Protein, Hemagglutinin, Adhesin

Growth Mode and Carbon Source Impact the Surfaceome Dynamics of Lactobacillus rhamnosus GG.

Bacterial biofilms have clear implications in disease and in food applications involving probiotics. Here, we show that switching the carbohydrate source from glucose to fructose increased the biofilm formation and the total surface-antigenicity of a well-known probiotic, Lactobacillus rhamnosus GG. Surfaceomes (all cell surface-associated proteins) of GG cells grown with glucose and fructose in planktonic and biofilm cultures were identified and compared, which indicated carbohydrate source-dependent variations, especially during biofilm growth. The most distinctive differences under these conditions were detected with several surface adhesins (e.g., MBF, SpaC pilus protein and penicillin-binding proteins), enzymes (glycoside hydrolases, PrsA, PrtP, PrtR, and HtrA) and moonlighting proteins (glycolytic, transcription/translation and stress-associated proteins, r-proteins, tRNA synthetases, Clp family proteins, PepC, PepN, and PepA). The abundance of several known adhesins and candidate moonlighters, including enzymes acting on casein-derived peptides (ClpP, PepC, and PepN), increased in the biofilm cells grown on fructose, from which the surface-associated aminopeptidase activity mediated by PepC and PepN was further confirmed by an enzymatic assay. The mucus binding factor (MBF) was found most abundant in fructose grown biofilm cells whereas SpaC adhesin was identified specifically from planktonic cells growing on fructose. An additional indirect ELISA indicated both growth mode- and carbohydrate-dependent differences in abundance of SpaC, whereas the overall adherence of GG assessed with porcine mucus indicated that the carbon source and the growth mode affected mucus adhesion. The adherence of GG cells to mucus was almost completely inhibited by anti-SpaC antibodies regardless of growth mode and/or carbohydrate source, indicating the key role of the SpaCBA pilus in adherence under the tested conditions. Altogether, our results suggest that carbon source and growth mode coordinate mechanisms shaping the proteinaceous composition of GG cell surface, which potentially contributes to resistance, nutrient acquisition and cell-cell interactions under different conditions. In conclusion, the present study shows that different growth regimes and conditions can have a profound impact on the adherent and antigenic features of GG, thereby providing new information on how to gain additional benefits from this probiotic.