Virulence Proteins Research Articles

Sodium lignosulfonate inhibits multiple virulent proteins of human fungal pathogen Candida albicans.

Systemic mycoses, particularly those caused by Candida albicans, represent a serious global health concern due to rising multidrug resistance and limited treatment options. This study explores the antifungal potential of sodium lignosulfonate (LIG), a natural phenolic compound, as a multitarget therapeutic agent against various virulence proteins of C. albicans and other pathogenic Candida species. The objective of this study was to further evaluate its multiple-targeting/polypharmacological potential with plausible mode of action against C. albicans. At first, LIG was subjected to in-silico analysis to acquire preliminary knowledge about its multiple targeting potential. Subsequently, some biochemical analyses were performed to demonstrate its fungicidal activity. In-vitro analysis (plasma membrane permeation, ROS production, chitin depletion study) was performed to further validate its promising multiple-targeting/polypharmacological potential and revealed its mechanism of action. Homology modeling and docking studies revealed that LIG effectively binds to critical C. albicans proteins, including ERG1, ERG11, FKS1, CHS3, CLB2, and CEK1. The docking scores indicated strong interactions, supporting LIG's potential to inhibit multiple virulence factors With ROS production we could confirm the involvement of apoptosis. Time-kill assays confirmed the antifungal effect of LIG against C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis. LIG demonstrated a >3-log10 reduction in CFU/mL, and in combination with fluconazole, it showed synergistic activity, particularly reducing CFU in C. dubliniensis by 2.5-fold compared to fluconazole alone. The chitin depletion assay has reported a decrease in levels of chitin which indicates another aspect of LIG's mode of action. This study reveals LIG as a potent and persuasive natural antifungal agent that targets multiple proteins of Candida. This revelation might impact the direction of potent antifungal agent development by aiming multiple targets of fungal pathogens simultaneously.

Mycobacterium tuberculosis Rv1048c affects the biological characteristics of recombinant Mycobacterium smegmatis

Tuberculosis is a serious, infectious, zoonotic disease caused by Mycobacterium tuberculosis. Infections are transmitted in humans and livestock via aerosols. Rv1048c is a hypothetical unknown protein in the standard strain of Mycobacterium tuberculosis H37Rv. Rv1048c exists only in pathogenic Mycobacterium tuberculosis and is highly conserved; however its function is still unclear. The recombinant Mycobacterium smegmatis strain Ms_Rv1048c, with heterologous expression of the Rv1048c gene, was constructed by using the pMV261 expression plasmid. The biological characteristics of the recombinant bacteria were studied, such as their growth pattern, drug resistance, and virulence. Expression of Rv1048c significantly reduced the growth rate of the strain, enhanced its ability to form a biofilm, and reduced its tolerance to sodium dodecyl sulfate, H2O2, and various anti-tuberculosis drugs, and reduced the viability of infected RAW264.7 macrophages. Rv1048c also significantly reduced the level of early pro-inflammatory factors in infected RAW264.7 cells. Rv1048c protein is considered to be a virulence protein that might regulate the growth of M. tuberculosis strains. The results of the present study indicate that Rv1048c plays an important role in Mycobacterial infection.

Broadly inhibitory antibodies to severe malaria virulence proteins.

Malaria pathology is driven by the accumulation of Plasmodium falciparum-infected erythrocytes in microvessels1. This process is mediated by the polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins of the parasite. A subset of PfEMP1 variants that bind to human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis2. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here we describe two broadly reactive and inhibitory human monoclonal antibodies to CIDRα1. The antibodies isolated from two different individuals exhibited similar and consistent EPCR-binding inhibition of diverse CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins, as well as parasite sequestration in bioengineered 3D human brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with three different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies are likely to represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

A translocation-competent pore is required for Shigella flexneri to escape from the double membrane vacuole during intercellular spread.

The type 3 secretion system (T3SS) is required for virulence in many bacterial pathogens that infect humans. The T3SS forms a pore through which virulence proteins are delivered into host cells to enable bacterial infection. Our work investigates the Shigella translocon pore protein IpaC, which is essential not only for bacteria to invade cells, but also for bacteria to spread between cells. An ability to spread between cells is essential for pathogenesis, thus understanding the mechanisms that enable spread is important for understanding how S. flexneri infection causes illness. We show that IpaC delivers virulence factors across the host membrane for S. flexneri to efficiently spread. This study furthers our understanding of the mechanisms involved in T3SS secretion and of translocon pore function during S. flexneri intercellular spread.

Comparative analysis of whole-genome sequences of African swine fever virus (Asfarviridae: Asfivirus) isolates сollected on the territory of the left bank of the Dnieper River in 2023.

The lack of data on the whole-genome sequences of African swine fever virus (ASFV) variants circulating on the territory of the left bank of the Dnieper River complicates the understanding of the molecular evolution of the virus and the character of the epidemic process development in Russia and Ukraine. Understanding the genetic divergence and phylogenetic relatedness of isolates can largely adjust the strategy of general and specific prevention of the disease. The aim of the study - search and description of unique mutations (deletions/insertions/substitutions) in isolates collected from domestic pigs in Donetsk, Luhansk and Zaporozhye regions in 2023; determination of relatedness and level of homology with reference strains of ASFV genotype II; sub-genotyping and clustering of isolates based on whole-genome analysis. The samples used were a culture suspension of porcine bone marrow (PBM) cells containing ASFV isolates obtained from pathologic material from domestic pig carcasses. Genomic DNA was prepared by purification and concentration of virus followed by phenol-chloroform extraction of total nucleic acid. The high-throughput sequencing process was performed using MGI technology. Consensus sequences were assembled by mapping reads to the reference genome of strain Georgia 2007/1. All isolates are assigned to genotype II, have a monophyletic origin, are phylogenetically close to the clusters «Europe» (4/5) and «Bryansk 2021» (1/5), and are divergent from the original parental genetic variants that make up the enlarged clades. In addition, numerous substitutions in the loci of the multigene family MGF 110, 505, and 360, encoding virulence proteins, were detected in 4 isolates from Donetsk and Zaporozhye regions. The phylogeny of the genotype II ASFV, which originated from the reference strain Georgia 2007/1, is shown to be sufficient for isolate differentiation. The presented data are of theoretical and practical importance for domestic and international ASFV surveillance.

Complete genome of single locus sequence typing D1 strain Cutibacterium acnes CN6 isolated from healthy facial skin.

Cutibacterium acnes is a Gram-positive bacterium commonly found on human skin, particularly in sebaceous areas. While it is typically considered a commensal, specific strain types based on single locus sequence typing (SLST) have been associated with pathogenic conditions or healthy skin. Recently, SLST D1 strains, part of phylotype IA1, have received attention for their potential benefits related to skin health. However, their genetic characteristics remain underexplored. Therefore, the whole genome of C. acnes CN6, an SLST D1 strain isolated from the facial skin of a healthy individual, was sequenced to expand the understanding of SLST D1 strains and identify genomic features that may support skin health. The whole genome sequencing of C. acnes CN6 was conducted using MinION reads based on de novo assembly, revealing a single circular complete chromosome. With the length of 2,550,458bp and G + C content of 60.04%, the genome contains 2,492 genes, including 2,433 CDSs, 9 rRNAs, 46 tRNAs, 4 ncRNAs, and 134 pseudo genes. Previously predicted virulence proteins of C. ances were detected in the genome. Genome comparation with 200 C. acnes strains isolated from healthy facial skin revealed SLST D1 strain-specific genes and a unique variant of the znuC gene in D1 strains.

Alginolyticus Virulence: An Overview

Introduction: Marine habitats harbor the gram-negative, halophilic Vibrio alginolyticus. Usually connected with aquatic life, it poses a serious health risk to humans, especially seafood and marine workers. Marine economic animals like prawns, fish, and shellfish can develop serious illnesses with rapid progression and significant fatality rates. Pathogenic relatives such as Vibrio cholerae and Vibrio parahaemolyticus, which carry genes for several virulence factors, have inherited its virulence, thereby increasing its clinical importance. No effective treatments exist to stop V. alginolyticus dissemination and infection; therefore, studying its virulence mechanisms is crucial to understanding its pathogenicity and improving prevention and therapy. Objectives: To analyze the virulence factors and antibiotic resistance mechanisms of Vibrio alginolyticus to improve prevention and control strategies for managing infections in aquaculture and public health settings. Methods: This study is a literature review synthesising findings from recent research on V. alginolyticus. Sources include peer-reviewed articles, clinical studies, and microbiological research focusing on virulence factors and antibiotic resistance. Data collection involved searching databases like PubMed, Scopus, and Web of Science, filtering for studies on V. alginolyticus’s virulence mechanisms, antibiotic resistance, and impacts on public health and aquaculture. We selected key studies to gain a comprehensive understanding of the pathogenic characteristics and resistance profiles of the bacterium. Results: The results show that V. alginolyticus uses many ways to be strong, like quorum sensing, making virulence proteins, secreting haemolysin, and making biofilms, which make it more dangerous to humans and marine animals. Several resistance genes and efflux systems contribute to its antibiotic resistance, thereby impeding the effectiveness of treatment. Studies also show that biofilm formation and quorum sensing facilitate survival in hostile environments, making infections more challenging to manage. Conclusions: Understanding the virulence factors and antibiotic resistance mechanisms of Vibrio alginolyticus is crucial for developing effective control strategies. This bacterium’s rapid spread and severe impact on marine life and human health necessitate new approaches in aquaculture management and clinical interventions. Enhanced monitoring and targeted research are essential to curb its spread and prevent infection, ultimately improving aquaculture productivity and reducing public health risks associated with this pathogen.

Identification and Characterization of a Novel Protein Drug Target WP_145928235.1 in Providencia Through Subtractive Proteomics and Molecular Dynamics Simulations

AbstractProvidencia stuartii, the most prevalent strain in its genus, is a significant cause of a range of potentially fatal diseases involving urinary tract infections, lung diseases, and endocarditis, often exhibiting resistance to multiple antibiotics, necessitating alternative therapeutic approaches. Unlike previous efforts focused on known virulence factors, this study delves into the physicochemical and functional characterization of 72 essential hypothetical proteins (EsHPs) from the P. stuartii BE246 genome. Subcellular localization identified 47 cytoplasmic EsHPs as potential drug targets, with the remainder 25 identified as vaccine targets. Subtractive analysis uncovered 68 virulent proteins, 4 of which were nonhomologous to both host and gut microbiota proteomes. Among these, WP_145928235.1 (147 amino acids), a cytoplasmic EsHP, emerged as a novel drug target due to its homologous broad‐spectrum hit. WP_145928235.1 displayed an instability index (II) of 33.14, an aliphatic index of 92.79, and the GRAVY score was ‐0.065 (hydrophilic). Molecular dynamics simulation‐aided conformational study and secondary structure analysis highlighted the protein's flexibility and propensity to interact with inhibitors. Key residues in the 60–90 region, including Asn62, Gly75, Glu81, and Asp85, were critical for interactions and stability, with low frustration indicating residual conservancy. WP_145928235.1 presents a promising target for therapeutic intervention against multidrug‐resistant P. stuartii infections.

Characteristics of vhvp-2 gene distribution and diversity within the Vibrio causing translucent post-larvae disease (TPD)

Translucent post-larva vibriosis (VTPD) in Penaeus vannamei, caused by the virulence factor Vibrio high virulent protein 2 (vhvp-2) gene, poses a significant threat to the shrimp aquaculture industry in China. This study conducted a comprehensive analysis of the vhvp-2 gene distribution and genetic diversity among Vibrio, which play a key role in the pathogenesis of translucent post-larvae disease (TPD). A total of 6281 Vibrio genomes from the NCBI database were screened, revealing the presence of the vhvp-2 gene in 26 strains, predominantly in V. parahaemolyticus, with diverse locations on both plasmids and chromosomes. Comparative analysis of the genomes, plasmids, and the vhvp-2 gene successfully differentiated three distinct clusters of Vibrio species and identified at least two major plasmid groups. However, these plasmid groups did not exhibit the same evolutionary relationships as the host bacteria themselves. The analysis also revealed the conserved presence of the vhvp-2 gene along with genetic variations. Moreover, the widespread presence of antimicrobial resistance genes (ARGs) and virulence factors in Vibrio strains enhances their pathogenicity and poses increased public health risks. The findings underscore the importance of understanding the adaptability, evolution, and ecological characteristics of Vibrio strains harboring vhvp-2 gene. This research provides a scientific foundation for the development of effective disease prevention and control strategies, contributing to the mitigation of TPD’s impact on the shrimp aquaculture industry and supporting its sustainable and healthy growth.

Helicobacter pylori biofilm interference by N-acyl homoserine lactonases: in vitro and in silico approaches.

Qurom quenching enzyme have an impact on treatment efficacy and prevent the recurrence of Helicobacter pylori biofilm-related infections, although it has not been thoroughly investigated in vitro and in silico. The current study aims to characterize the N-acyl homoserine lactonase, the quorum quenching AiiA protein of Bacillus licheniformis against H. pylori biofilm. In this study, AiiA protein were screened for their anti-biofilm activity, was found to effectively control biofilm formation of H. pylori with concentrations ranging from 2 to 10µg/mL. According to CLSM and COMSTAT analysis, the untreated substratum had the robust biofilm biomass of 25-18 µM and biovolume of 3-4 mm3 /mm2. The total biofilm biovolume and average biofilm thickness were considerably reduced by 40% with a single application of 10µg/mL of AiiA protein. The biofilm treated with AiiA exhibited a lower urease and polysaccharides than to the untreated biofilm. Further, in silico analysis, exhibited a greater interaction of AiiA against the outer membrane proteins of H. pylori compared to virulence factors. The conserved domains in the binding pockets of AiiA proteins showed a highest binding affinity proving the catalytic activity of the protein. In this study, the H. pylori biofilm architecture, exopolysaccharide and urease were significantly controlled by our purified N-acyl homoserine lactonase from B. licheniformis. Furthermore, the molecular docking showed the significant interaction between AiiA and key biofilm forming and virulence proteins proved an excellent antibiofilm activity controlling the infections of H. pylori human pathogen.

The pathogenic responses elicited during exposure of human intestinal cell line with Giardia duodenalis excretory-secretory products and the potential attributed endocytosis mechanism.

Giardia duodenalis, an important zoonotic protozoan parasite, adheres to host intestinal epithelial cells (IECs) via the ventral disc and causes giardiasis characterized mainly by diarrhea. To date, it remains elusive how excretory-secretory products (ESPs) of Giardia enter IECs and how the cells respond to the entry. Herein, we initially demonstrated that ESPs evoked IEC endocytosis in vitro. We indicated that ESPs contributed vitally in triggering intrinsic apoptosis, pro-inflammatory responses, tight junction (TJ) protein expressional changes, and autophagy in IECs. Endocytosis was further proven to be implicated in those ESPs-triggered IEC responses. Ten predicted virulent excretory-secretory proteins of G. duodenalis were investigated for their capability to activate clathrin/caveolin-mediated endocytosis (CME/CavME) in IECs. Pyridoxamine 5'-phosphate oxidase (PNPO) was confirmed to be an important contributor. PNPO was subsequently verified as a vital promoter in the induction of giardiasis-related IEC apoptosis, inflammation, and TJ protein downregulation. Most importantly, this process seemed to be involved majorly in PNPO-evoked CME pathway, rather than CavME. Collectively, this study identified Giardia ESPs, notably PNPO, as potentially important pathogenic factors during noninvasive infection. It was also noteworthy that ESPs-evoked endocytosis might play a role in triggering giardiasis-inducing cellular regulation. These findings would deepen our understanding about the role of ESPs, notably PNPO, in the pathogenesis of giardiasis and the potential attributed endocytosis mechanism.

Nanocatalysis of silver-nanobioprobe based supersensitive electrochemical detection of Salmonella serotypes targeting virulence protein

Herein, we report a supersensitive and specific detection of Salmonella employing nanocatalysis of silver nanoparticle (AgNp). A nanobioprobe was developed employing specific antibody (Ab) that binds to a peptide present in transmembrane protein of Salmonella. We have studied 7 surface-exposed peptide hits from conserved virulence proteins (PagC, ST50, PagN, CdtB and FliC). These peptides were experimentally evaluated by BLI (Bio layer interferometry) for their reactivity towards antisera raised against an admix of major Salmonella serogroups. The most promising peptide was used to generate Ab with binding affinity Kd of 5.6 × 10−9 M. The Ab exhibited high specificity towards entire Salmonella serotypes prevalent in foods, as illustrated by FACS (Fluorescence-activated cell sorting) study. The Ab-AgNp probe was blocked with a dual layer to prevent non-specific interactions, confirmed by employing BLI and TEM (Transmission electron microscopy). For the electrochemical detection, the autonanocatalysis of AgNp in presence of H2O2 was used to generate numerous Ag+ resulting in an amplified signal that could detect 10 cells/mL. The relative standard deviation (RSD) was observed to be 4.5%. The platform achieved recovery of 100–112% calculated for 102 cells/mL. The performance was validated in milk, buffer peptone water (BPW) and tap water by spiking studies. The study highlights the effectiveness of efficiently blocked AgNp-mediated probes for the highly selective and sensitive detection of Salmonella, representing a significant advancement in bacterial sensing.

Using reverse vaccinology techniques combined with B-cell epitope prediction to screen potential antigenic proteins of the bovine pathogen Clostridium perfringens type A

Clostridium perfringens type A frequently causes necrohaemorrhagic enteritis in cattle, a rapidly progressing disease with a high mortality rate, thus inflicting substantial economic losses in the cattle industry. Effective prevention and control of this disease rely on rapid detection and vaccination strategies, making the screening of antigenic proteins with diagnostic and vaccine potential particularly crucial. In this study, we conducted a pangenomic analysis of 15 bacterial strains, grounded in traditional reverse vaccinology and supplemented with B-cell linear and conformational epitope analysis tools. This approach led to the identification of 2304 core genes and 3606 accessory genes, among which 58 surface-exposed proteins, encoded by core genes, were identified Proteins lacking tertiary structure information were predicted via AlphaFold2, ultimately identifying four target proteins and 14 candidate proteins enriched with linear and conformational epitopes, including virulence proteins such as alpha-toxin, theta-toxin, and alpha-clostripain, and extracellular solute-binding proteins, rhodanese-like proteins, and the accessory gene-encoded lysozyme inhibitor LprI family protein. Our findings demonstrate that the combined use of multiple B-cell epitope analysis tools can help overcome the limitations of any single tool. The proteins selected in this study offer valuable references for rapid diagnostics and the development of genetically engineered vaccines.

Agrobacterium virulence factors induce the expression of host DNA repair-related genes without promoting major genomic damage

This study aimed to investigate whether the plant DNA damage levels and DNA damage response (DDR) are regulated during Agrobacterium infection and potentially manipulated by Agrobacterium to facilitate T-DNA integration. We investigated the plant genomic response to Agrobacterium infection by measuring gamma H2AX levels, which reflect the levels of double-strand DNA breaks (DSBs), and by characterizing transcription of three major DNA repair marker genes NAC82, KU70, and AGO2. These experiments revealed that, globally, Agrobacterium infection did not result in a major increase in DSB content in the host genome. The transcription of the DNA damage repair genes, on the other hand, was elevated upon the wild-type Agrobacterium infection. This transcriptional outcome was largely negated by a mutation in the bacterial virB5 gene which encodes the virulence (Vir) protein B5, a minor component of Agrobacterium pilus necessary for the translocation of Vir effector proteins into the host cell, suggesting that the transcriptional activation of the cellular DNA damage repair machinery requires the transport into the host cell of the Agrobacterium effectors, i.e., the VirD2, VirD5, VirE2, VirE3, and VirF proteins. Most likely, a combination of several of these Vir effectors is required to activate the host DNA repair as their individual loss- or gain-of-function mutants did not significantly affect this process.

Unveiling Insights into the Whole Genome Sequencing of Mycobacterium spp. Isolated from Siamese Fighting Fish (Betta splendens)

This study aims to genomically elucidate six isolates of rapidly growing non-tuberculous mycobacteria (RGM) derived from Siamese fighting fish (Betta splendens). These isolates had previously undergone phenotypic and biochemical characterization, antibiotic susceptibility testing, and in vivo virulence assessment. Initial DNA barcoding using the 16S rRNA sequence assigned these six isolates to five different species, namely Mycobacterium chelonae (BN1983), M. cosmeticum (BN1984 and N041), M. farcinogenes (SNSK5), M. mucogenicum (BN1956), and M. senegalense (BN1985). However, the identification relied solely on the highest percent identity of the 16S rRNA gene, raising concerns about the taxonomic ambiguity of these species. Comprehensive whole genome sequencing (WGS) and extended genomic comparisons using multilocus sequence typing (MLST), average nucleotide identity (ANI), and digital DNA–DNA hybridization (dDDH) led to the reclassification of BN1985 and SNSK5 as M. conceptionense while confirming BN1983 as M. chelonae and BN1984 and N041 as M. cosmeticum. Notably, the analysis of the BN1956 isolate revealed a potential new species that is proposed here as M. mucogenicum subsp. phocaicum sp. nov. Common genes encoding “mycobacterial” virulence proteins, such as PE and PPE family proteins, MCE, and YrbE proteins, were detected in all six isolates. Two species, namely M. chelonae and M. cosmeticum, appear to have horizontally acquired T6SS-II (clpB), catalase (katA), GroEL (groel), and capsule (rmlb) from distantly related environmental bacteria such as Klebsiella sp., Neisseria sp., Clostridium sp., and Streptococcus sp. This study provides the first draft genome sequence of RGM isolates currently circulating in B. splendens and underscores the necessity of WGS for the identification and classification of mycobacterial species.

Overexpression, purification and biochemical studies of Sortase A from Enterococcus faecalis (Ef) and its inhibition studies with Aloenin

Sortase A (SrtA) is a bacterial transpeptidase that garnishes the bacterial surface by adding various virulent factors or proteins by cleaving the LPXTG-specific motif between T and G amino acids. These virulence factors assist in the attachment of host cells, which are essential for bacterial virulence. Enterococcus species are among the multidrug-resistant bacteria that cause nosocomial infections; they have drawn a lot of attention recently. SrtA from E. faecalis (Ef) plays a critical role in pathogenesis, making it a suitable target for the development of antibacterial agents. Since SrtA is not involved in bacterial growth and is present on the surface of bacteria, the probability of developing antibiotic resistance is minimal. In this work, we have done cloning, expression and purification of Ef-SrtA using IMAC (Immobilised Metal Affinity Chromatography) followed by Gel filtration chromatography. Purified Ef-SrtA showed maximum activity at pH-8 and temperature between 45 and 55 °C. The fluorescent assay for kinetic studies of Ef-SrtA showed Vmax 3.852 µM.min−1 and kcat 7.7 × 10−2s−1 for the hydrolysis of substrate using Abz-LPETG-K(Dnp)-NH2. We have selected fifteen Aloe vera extracted compounds and performed virtual screening and docking experiments to identify potential inhibitors against Ef-SrtA. Among fifteen molecules, Aloenin-a which was bound to the active site with a binding energy of -6.1 kcal/mol, interacted with the active site residues, Arg139, Pro105, Leu39, Ala46, and Cys126. Aloenin-a showed a significant inhibitory effect against Ef-SrtA, with an IC50 value of 20.68 µM. Aloenin-a inhibits biofilm formation at concentrations of 20–250 µg/mL. The fibrinogen assay showed adherence to fibrinogen was reduced in the presence of Aloenin-a for E. faecalis. The results demonstrated that Aloe vera extracts containing Aloenin-a can be a significant antagonist of Ef-SrtA.

Plasmonic-Enhanced Colorimetric Lateral Flow Immunoassays Using Bimetallic Silver-Coated Gold Nanostars.

The colorimetric lateral flow immunoassay (cLFIA) has gained widespread attention as a point-of-care testing (POCT) technique due to its low cost, short analysis time, portability, and capability of being performed by unskilled operators with minimal requirement of reagents. However, the low analytical sensitivity of conventional LFIA based on colloidal gold nanospheres limits their applications for sensitive detection of trace amounts of target analytes. In this study, we introduced a novel plasmonic-enhanced colorimetric LFIA (PE-cLFIA) platform featuring bimetallic silver-coated gold nanostars (BGNS) with exceptional optical properties, leading to ultrahigh visual color brightness. The BGNS-based PE-cLFIA was successfully applied to detect a model analyte, low-calcium response V (LcrV), a virulence protein factor found in Yersinia pestis, the causative agent of bubonic plague. The PE-cLFIA sensing using BGNS-3 composed of 45 nm silver thickness showed a high visual colorimetric sensitivity with a detection limit as low as 13.7 pg/mL, which was around 50 times more sensitive than that of a traditional gold nanoparticle-based LFIA. In addition, the antibody-conjugated BGNS-3 showed excellent stability over 6 months. To illustrate the potential for clinical applications, we demonstrated that our LFIA platform for detecting LcrV spiked in human serum without any sample preprocessing exhibited a detection limit of 22.8 pg/mL. These results open up new opportunities for developing hybrid nanoparticle systems for sensitive POCT PE-cLFIA screening for infectious disease detection.

Metagenomic analysis of pathogenic bacteria and virulence factor genes in coastal sediments from highly urbanized cities of India

A metagenomic approach was employed to investigate the diversity and distribution of Virulence Factors Genes (VFGs) and Pathogenic Bacteria (PB) in sediment samples collected from highly urbanized cities along the Indian coastline. Among the study locations, Mumbai, Veraval and Paradeep showed a higher abundance of PB, with Vibrio and Pseudomonas as dominant at the genus level, and Escherichia coli and Pseudomonas aeruginosa at the species level. In total, 295 VFGs were detected across all sediment samples, of which 40 VFGs showed a similarity of ≥90 % with the Virulence Database (VFDB) and were focused in this study. Among the virulent proteins, twitching motility protein and flagellar P-ring were found to be prevalent and significantly associated with Vibrio spp., and Pseudomonas spp., indicating potential bacterial pathogenicity. This investigation serves as the basis for future studies and provides insights into the comprehensive taxonomic profiles of PB, VFGs and their associated PB in the coastal sediments of India.

First crystal structure of the DUF2436 domain of virulence proteins from Porphyromonas gingivalis.

Porphyromonas gingivalis is a major pathogenic oral bacterium that is responsible for periodontal disease. It is linked to chronic periodontitis, gingivitis and aggressive periodontitis. P. gingivalis exerts its pathogenic effects through mechanisms such as immune evasion and tissue destruction, primarily by secreting various factors, including cysteine proteases such as gingipain K (Kgp), gingipain R (RgpA and RgpB) and PrtH (UniProtKB ID P46071). Virulence proteins comprise multiple domains, including the pro-peptide region, catalytic domain, K domain, R domain and DUF2436 domain. While there is a growing database of knowledge on virulence proteins and domains, there was no prior evidence or information regarding the structure and biological function ofthe well conserved DUF2436 domain. In this study, the DUF2436 domain of PrtH from P. gingivalis (PgDUF2436) was determined at 2.21 Å resolution, revealing a noncanonical β-jelly-roll sandwich topology with two antiparallel β-sheets and one short α-helix. Although the structure of PgDUF2436 was determined by the molecular-replacement method using an AlphaFold model structure as a template, there were significant differences in the positions of β1 between the AlphaFold model and the experimentally determined PgDUF2436 structure. The Basic Local Alignment Search Tool sequence-similarity search program showed no sequentially similar proteins in the Protein Data Bank. However, DaliLite search results using structure-based alignment revealed that the PgDUF2436 structure has structural similarity Z-scores of 5.9-5.4 with the C-terminal domain of AlgF, the D4 domain of cytolysin, IglE and the extracellular domain structure of PepT2. This study has elucidated the structure of the DUF2436 domain for the first time and a comparative analysis with similar structures has been performed.

Diversity, Distribution and Structural Prediction of the Pathogenic Bacterial Effectors EspN and EspS.

Many Gram-negative enterobacteria translocate virulence proteins (effectors) into intestinal epithelial cells using a type III secretion system (T3SS) to subvert the activity of various cell functions possess. Many T3SS effectors have been extensively characterized, but there are still some effector proteins whose functional information is completely unknown. In this study, two predicted effectors of unknown function, EspN and EspS (Escherichia coli secreted protein N and S), were selected for analysis of translocation, distribution and structure prediction. The TEM1 (β-lactamase) translocation assay was performed, which showed that EspN and EspS are translocated into host cells in a T3SS-dependent manner during bacterial infection. A phylogenetic tree analysis revealed that homologs of EspN and EspS are widely distributed in pathogenic bacteria. Multiple sequence alignment revealed that EspN and its homologs share a conserved C-terminal region (673-1133 a.a.). Furthermore, the structure of EspN (673-1133 a.a.) was also predicted and well-defined, which showed that it has three subdomains connected by a loop region. EspS and its homologs share a sequence-conserved C-terminal (146-291 a.a.). The predicted structure of EspS (146-291 a.a.) is composed of a β-sheet consisting of four β-strands and several short helices, which has a TM score of 0.5014 with the structure of the Vibrio cholerae RTX cysteine protease domain (PDBID: 3eeb). These results suggest that EspN and EspS may represent two important classes of T3SS effectors associated with pathogen virulence, and our findings provide important clues to understanding the potential functions of EspN and EspS.