Virulence Proteins Research Articles

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.

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.

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 x 10-9 M. The Ab exhibited high specificity towards entire Salmonella serotypes prevalent in foods, as illustrated by the 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.

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.

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.

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.

Genetic evidence for a regulated cysteine protease catalytic triad in LegA7, a Legionella pneumophila protein that impinges on a stress response pathway.

Legionella pneumophila grows in a membrane-bound compartment in macrophages during disease. Construction of the compartment requires a dedicated secretion system that translocates virulence proteins into host cells. One of these proteins, LegA7, is shown to activate a stress response pathway in host cells called the mitogen-activated protein kinase (MAPK) pathway. The effects on the mammalian MAPK pathway were reconstructed in yeast, allowing the development of a strategy to identify the role of individual domains of LegA7. A domain similar to cysteine proteases is demonstrated to be critical for impinging on the MAPK pathway, and the catalytic activity of this domain is required for targeting this path. In addition, a conserved series of repeats, called ankyrin repeats, controls this activity. Data are provided that argue the interaction of the ankyrin repeats with unknown targets probably results in activation of the cysteine protease domain.

EspH utilizes phosphoinositide and Rab binding domains to interact with plasma membrane infection sites and Rab GTPases*

ABSTRACT Enteropathogenic E. coli (EPEC) is a Gram-negative bacterial pathogen that causes persistent diarrhea. Upon attachment to the apical plasma membrane of the intestinal epithelium, the pathogen translocates virulence proteins called effectors into the infected cells. These effectors hijack numerous host processes for the pathogen’s benefit. Therefore, studying the mechanisms underlying their action is crucial for a better understanding of the disease. We show that translocated EspH interacts with multiple host Rab GTPases. AlphaFold predictions and site-directed mutagenesis identified glutamic acid and lysine at positions 37 and 41 as Rab interacting residues in EspH. Mutating these sites abolished the ability of EspH to inhibit Akt and mTORC1 signaling, lysosomal exocytosis, and bacterial invasion. Knocking out the endogenous Rab8a gene expression highlighted the involvement of Rab8a in Akt/mTORC1 signaling and lysosomal exocytosis. A phosphoinositide binding domain with a critical tyrosine was identified in EspH. Mutating the tyrosine abolished the localization of EspH at infection sites and its capacity to interact with the Rabs. Our data suggest novel EspH-dependent mechanisms that elicit immune signaling and membrane trafficking during EPEC infection.

A novel brominated chalcone derivative as a promising multi-target inhibitor against multidrug-resistant Listeria monocytogenes

Foodborne pathogens continue to challenge public health due to their ability to cause severe illness and their increasing resistance to current antimicrobial treatments. Listeria monocytogenes is a resilient foodborne pathogen that poses significant risks to vulnerable populations, leading to severe infections and high hospitalization rates. The emergence of antimicrobial-resistant (AMR) strains of L. monocytogenes underscores the need for novel therapeutic strategies. In this study, we investigated the antimicrobial efficacy of the (2E)-3-(3,5-dibromo-2-hydroxylphenyl)-1-(5-methylfuran-2-yl) prop-2-en-1-one (DK06) against multidrug-resistant L. monocytogenes. DK06 exhibited a significant dose-dependent inhibition of L. monocytogenes growth, achieving a maximum inhibition of 92.9 % at 320 μM. Molecular docking and dynamics simulations revealed high binding affinities for key virulence proteins PlcB and ArgA, with stable protein-ligand interactions. DK06 also disrupted biofilm formation at sub-MIC levels, reducing extracellular polymeric substances (EPS) and biofilm mass, as observed by scanning electron microscopy (SEM) analysis. Furthermore, DK06 downregulated the expression of virulence genes (plcB, argA, and hly) and decreased hemolytic activity. In vivo zebrafish studies confirmed the safety of DK06 up to 80 μM, demonstrating its efficacy in reducing mortality and oxidative stress associated with L. monocytogenes infection. DK06 also attenuated inflammation by downregulating key inflammatory markers (tnfa, il1b, il6, and nfkb). These findings indicate that DK06 is a promising multi-target inhibitor with potential application in treating infections and combating antimicrobial resistance.

Temperature-dependent alterations in the proteome of the emergent fish pathogen Edwardsiella piscicida.

Edwardsiella piscicida is an emerging bacterial pathogen and the aetiological agent of edwardsiellosis among cultured and wild fish species globally. The increased frequency of outbreaks of this Gram-negative, facultative intracellular pathogen pose not only a threat to the aquaculture industry but also a possible foodborne/waterborne public health risk due to the ill-defined zoonotic potential. Thus, understanding the role of temperature on the virulence of this emerging pathogen is essential for comprehending the pathogenesis of piscine edwardsiellosis in the context of current warming trends associated with climate change, as well as providing insight into its zoonotic potential. In this study, significant temperature-dependent alterations in bacterial growth patterns were observed, with bacterial isolates grown at 17°C displaying higher peak growth sizes, extended lag times, and slower maximal growth rates than isolates grown at 27or 37°C. When E. piscicida isolates were grown at 37°C compared to 27 and 17°C, mass spectrometry analysis of the E. piscicida proteome revealed significant downregulation of crucial virulence proteins, such as Type VI secretion system proteins and flagellar proteins. Although invivo models of infection are warranted, this invitro data suggests possible temperature-associated alterations in the virulence and pathogenic potential of E. piscicida in poikilotherms and homeotherms.

Interferon lambda signaling in neutrophils enhances the pathogenesis of Bordetella pertussis infection

Abstract Interferon lambda plays diverse roles in bacterial infections. Previously, we showed that interferon lambda is induced in the lungs of Bordetella pertussis-infected adult mice and exacerbates inflammation. Here, we report that mice lacking the interferon lambda receptor 1 specifically on neutrophils (MRP8creIFNLR1fl/fl mice) exhibit reduced lung bacterial load and inflammation compared to wild-type mice during B. pertussis infection. In B. pertussis-infected wild-type mice, lung type I and III IFN responses were higher than in MRP8creIFNLR1fl/fl mice, correlating with increased lung inflammatory pathology. There was an increased proportion of interferon gamma-producing neutrophils in the lungs of MRP8creIFNLR1fl/fl mice compared to wild-type mice. IFNLR1−/− neutrophils incubated with B. pertussis exhibited higher killing compared to wild-type neutrophils. Treatment of wild-type neutrophils with interferon lambda further decreased their bacterial killing capacity and treatment of wild-type mice with interferon lambda increased lung bacterial loads. Contributing to the differential killing, we found that IFNLR1−/− neutrophils exhibit higher levels of reactive oxygen species, myeloperoxidase, matrix metalloproteinase-9 activity, neutrophil extracellular traps, and interferon gamma secretion than wild-type neutrophils, and inhibiting NADPH oxidase inhibited bacterial killing in IFNLR1−/− neutrophils. B. pertussis-induced interferon lambda secretion and IFNLR1 gene expression in mouse and human neutrophils and this was dependent on the bacterial virulence protein pertussis toxin. Pertussis toxin enhanced bacterial loads in wild type but not in MRP8creIFNLR1fl/fl or IFNLR1−/− mice. Thus, pertussis toxin disrupts neutrophil function by enhancing type III IFN signaling, which prevents neutrophils from effectively clearing B. pertussis during infection, leading to higher bacterial loads and exacerbation of lung inflammation.

In silico analysis of secreted effectorome of the rubber tree pathogen Rigidoporus microporus highlights its potential virulence proteins.

Rigidoporus microporus, the causative agent of the white root rot disease of rubber trees, poses a significant threat to natural rubber production worldwide. Understanding the molecular mechanisms facilitating its pathogenicity would be crucial for developing effective disease management strategies. The pathogen secretes effector proteins, which play pivotal roles in modulating host immune responses and infection. In this study, in silico analyses identified 357 putative secreted effector proteins from the R. microporus genome. These were then integrated into previous RNA-seq data obtained in response to rubber tree latex exposure. Annotation of putative effectors suggested the abundance of proteins in several families associated with the virulence of R. microporus, especially hydrophobin proteins and glycoside hydrolase (GH) proteins. The contribution of secreted effectors to fungal pathogenicity was discussed, particularly in response to rubber tree latex exposure. Some unknown highly expressed effectors were predicted for the protein structures, revealing their similarity to aminopeptidase, ubiquitin ligase, spherulin, and thaumatin protein. This integrative study further elucidates the molecular mechanism of R. microporus pathogenesis and offers alternative targets for developing control strategies for managing white root rot disease in rubber plantations.

Comparison of Mycobacterium tuberculosis Strains with H37Rv Using Whole Genome Sequence Analysis to Identify Virulence Factors and Phylogenetic Relationships

Mycobacterium tuberculosis (Mtb) poses a significant disease burden in developing countries, largely due to the rising issue of drug resistance. In Pakistan, the genetic and molecular characteristics of resistant strains have not been thoroughly analyzed or compared with the reference Mtb strain, H37Rv, to understand the molecular and genetic epidemiology. Mtb employs various strategies to resist anti-tuberculosis drugs, and antimicrobial resistance, including single and multidrug resistance, has increased extensively in developing nations like Pakistan. In this study, the whole genomes of three Mtb strains (335, 335-2, and 311) were uploaded to the Rapid Annotation Subsystems Technology (RAST) server to retrieve coding DNA sequences (CDS). The RAST server identified 4,454, 4,445, and 4,396 CDS for Mtb strains 335, 335-2, and 311, respectively. Mutation analysis was performed using NCBI nucleotide BLAST and CLUSTAL Omega, comparing the sequences with the H37Rv reference strain. The analysis revealed 909, 832, and 1,135 mutations in the 335, 335-2, and 311 strains, respectively. Phylogenetic analysis based on the most virulent protein (EccC3) indicated a close resemblance between all three strains and the H37Rv strain. Furthermore, Mycobacteriumcanettii (accession number WP_203037448.1) was identified as the closest related bacterium to the studied strains and H37Rv.

Oral inoculation of Fusobacterium nucleatum exacerbates ulcerative colitis via the secretion of virulence adhesin FadA

ABSTRACT Fusobacterium nucleatum (F. nucleatum), an anaerobic resident of the oral cavity, is increasingly recognized as a contributing factor to ulcerative colitis (UC). The adhesive properties of F. nucleatum are mediated by its key virulence protein, FadA adhesin. However, further investigations are needed to understand the pathogenic mechanisms of this oral pathogen in UC. The present study aimed to explore the role of the FadA adhesin in the colonization and invasion of oral F. nucleatum in dextran sulphate sodium (DSS)-induced colitis mice via molecular techniques. In this study, we found that oral inoculation of F. nucleatum strain carrying the FadA adhesin further exacerbated DSS-induced colitis, leading to elevated alveolar bone loss, disease severity, and mortality. Additionally, CDH1 gene knockout mice treated with DSS presented increases in body weight and alveolar bone density, as well as a reduction in disease severity. Furthermore, FadA adhesin adhered to its mucosal receptor E-cadherin, leading to the phosphorylation of β-catenin and the degradation of IκBα, the activation of the NF-κB signalling pathway and the upregulation of downstream cytokines. In conclusion, this research revealed that oral inoculation with F. nucleatum facilitates experimental colitis via the secretion of the virulence adhesin FadA. Targeting the oral pathogen F. nucleatum and its virulence factor FadA may represent a promising therapeutic approach for a portion of UC patients.

Protein disulfide isomerase A4 binds to Brucella BtpB and mediates intracellular NAD+/NADH metabolism in RAW264.7 cells

The Toll/interleukin-1 receptor (TIR) signaling domain is distributed widely in mammalian Toll-like receptors and adaptors, plant nucleotide-binding leucine-rich repeat receptors, and specific bacterial virulence proteins. Proteins that possess TIR domain exhibit NADase activity which is distinct from the canonical signaling function of these domains. However, the effects of bacterial TIR domain proteins on host metabolic switches and the underlying mechanism of NADase activity in these proteins remain unclear. Here, we utilized Brucella TIR domain-containing type IV secretion system effector protein, BtpB, to explore the mechanism of NADase activity in host cells. We showed that using ectopic expression BtpB not only generates depletion of NAD+ but also loss of NADH and ATP in RAW264.7 macrophage cells. Moreover, immunoprecipitation-mass spectrometry, co-immunoprecipitation, and confocal microscope assays revealed that BtpB interacted with host protein disulfide isomerase A4 (PDIA4). The Brucella mutant strain deleted the gene for BtpB, significantly decreased PDIA4 expression. Furthermore, our data revealed that PDIA4 played an important role in regulating intracellular NAD+/NADH levels in macrophages, and PDIA4 overexpression restored the decline of intracellular NAD+ and NADH levels induced by Brucella BtpB. The results provide new insights into the metabolic regulatory activity of TIR domain proteins in the critical human and animal pathogen Brucella.

Arabidopsis F-box proteins D5BF1 and D5BF2 negatively regulate Agrobacterium-mediated transformation andtumorigenesis.

The pathogen Agrobacterium tumefaciens is known for causing crown gall tumours in plants. However, it has also been harnessed as a valuable tool for plant genetic transformation. Apart from the T-DNA, Agrobacterium also delivers at least five virulence proteins into the host plant cells, which are required for an efficient infection. One of these virulence proteins is VirD5. F-box proteins, encoded in the host plant genome or the Ti plasmid, and the ubiquitin/26S proteasome system (UPS) also play an important role in facilitating Agrobacterium infection. Our study identified two Arabidopsis F-box proteins, D5BF1 and D5BF2, that bind VirD5 and facilitate its degradation via the UPS. Additionally, we found that Agrobacterium partially suppresses the expression of D5BF1 and D5BF2. Lastly, stable transformation and tumorigenesis efficiency assays revealed that D5BF1 and D5BF2 negatively regulate the Agrobacterium infection process, showing that the plant F-box proteins and UPS play a role in defending against Agrobacterium infection.

Proteomic characterization of Tenacibaculum dicentrarchi under iron limitation reveals an upregulation of proteins related to iron oxidation and reduction metabolism, iron uptake systems and gliding motility.

A strategy for vaccine design involves identifying proteins that could be involved in pathogen-host interactions. The aim of this proteomic study was to determine how iron limitation affects the protein expression of Tenacibaculum dicentrarchi, with a primary focus on virulence factors and proteins associated with iron uptake. The proteomic analysis was carried out using two strains of T. dicentrarchi grown under normal (control) and iron-limited conditions, mimicking the host environment. Our findings revealed differences in the proteins expressed by the type strain CECT 7612T and the Chilean strain TdCh05 of T. dicentrarchi. Nonetheless, both share a common response to iron deprivation, with an increased expression of proteins associated with iron oxidation and reduction metabolism (e.g., SufA, YpmQ, SufD), siderophore transport (e.g., ExbD, TonB-dependent receptor, HbpA), heme compound biosynthesis, and iron transporters under iron limitation. Proteins involved in gliding motility, such as GldL and SprE, were also upregulated in both strains. A negative differential regulation of metabolic proteins, particularly those associated with amino acid biosynthesis, was observed under iron limitation, reflecting the impact of iron availability on bacterial metabolism. Additionally, the TdCh05 strain exhibited unique proteins associated with gliding motility machinery and phage infection control compared to the type strain. These groups of proteins have been identified as virulence factors within the Flavobacteriaceae family, including the genus Tenacibaculum. These results build upon our previous report on iron acquisition mechanisms and could lay the groundwork for future studies aimed at elucidating the role of some of the described proteins in the infectious process of tenacibaculosis, as well as in the development of potential vaccines.