Opportunistic Human Pathogen Research Articles

Deciphering Cronobacter sakazakii pathogenicity: Exploring virulence factors and host interactions": A short review

Opportunistic human pathogen called Cronobacter spp. can cause severe illnesses in immunocompromised individuals and newborns. These species are mostly responsible for disorders like meningitis, necrotizing enterocolitis, and bacteremia because they can survive in very dry foods like PIF and have also been identified in various environmental niches, including plants. These species' contamination is ascribed to biofilms, which are biotic or abiotic surface communities. Because they build biofilms on a variety of surfaces, identifying and eliminating them is extremely difficult. For the purpose of creating efficient treatment interventions and preventative measures, it is essential to comprehend the processes of virulence and the relationship between this pathogen and its host. A variety of virulence factors, such as adhesion factors, invasion proteins, biofilm formation components, and secreted toxins, contribute to Cronobacter spp. pathogenicity. Genes involved in capsule formation—such as those involved in exopolysaccharide synthesis—are essential to its pathogenicity because they are critical in desiccation resistance and resistance to host immunological defenses. Furthermore, genes that encode flagella, fimbriae, and outer membrane proteins are linked to host cell adherence and invasion. This review also explores the emerging link between Cronobacter spp. and plants, highlighting the bacterium's ability to survive and persist in the environment because Plants may act as reservoirs, contributing to the contamination of raw agricultural products, which can subsequently enter the food supply chain. Developing focused preventative measures and treatment approaches requires knowledge of the various biological as well as genetic factors that contribute to Cronobacter spp. virulence and how it affects the host. This review clarifies the effects of Cronobacter spp. on the host and investigates the genes linked to virulence in Cronobacter.

InSeq analysis of defined Legionella pneumophila libraries identifies a transporter-encoding gene cluster important for intracellular replication in mammalian hosts.

Legionella pneumophila is an intracellular bacterial pathogen that replicates inside human alveolar macrophages to cause a severe pneumonia known as Legionnaires' disease. L. pneumophila requires the Dot/Icm Type IV secretion system to deliver hundreds of bacterial proteins to the host cytosol that manipulate cellular processes to establish a protected compartment for bacterial replication known as the Legionella-containing vacuole. To better understand mechanisms apart from the Dot/Icm system that support survival and replication in this vacuole, we used transposon insertion sequencing in combination with defined mutant sublibraries to identify L. pneumophila fitness determinants in primary mouse macrophages and the mouse lung. This approach validated that many previously identified genes important for intracellular replication were critical for infection of a mammalian host. Further, the screens uncovered additional genes contributing to L. pneumophila replication in mammalian infection models. This included a cluster of seven genes in which insertion mutations resulted in L. pneumophila fitness defects in mammalian hosts. Generation of isogenic deletion mutants and genetic complementation studies verified the importance of genes within this locus for infection of mammalian cells. Genes in this cluster are predicted to encode nucleotide-modifying enzymes, a protein of unknown function, and an atypical ATP-binding cassette (ABC) transporter with significant homology to multidrug efflux pumps that has been named Lit, for Legionella infectivity transporter. Overall, these data provide a comprehensive overview of the bacterial processes that support L. pneumophila replication in a mammalian host and offer insight into the unique challenges posed by the intravacuolar environment.IMPORTANCEIntracellular bacteria employ diverse mechanisms to survive and replicate inside the inhospitable environment of host cells. Legionella pneumophila is an opportunistic human pathogen and a model system for studying intracellular host-pathogen interactions. Transposon sequencing is an invaluable tool for identifying bacterial genes contributing to infection, but current animal models for L. pneumophila are suboptimal for conventional screens using saturated mutant libraries. This study employed a series of defined transposon mutant libraries to identify determinants of L. pneumophila fitness in mammalian hosts, which include a newly identified bacterial transporter called Lit. Understanding the requirements for survival and replication inside host cells informs us about the environment bacteria encounter during infection and the mechanisms they employ to make this environment habitable. Such knowledge will be key to addressing future challenges in treating infections caused by intracellular bacteria.

Bispecific Thio-linked Disaccharides as Inhibitors of Pseudomonas aeruginosa Lectins LecA (PA-IL) and LecB (PA-IIL): Dual-Targeting Strategy.

Pseudomonas aeruginosa is a prevalent opportunistic human pathogen, particularly associated with cystic fibrosis. Among its virulence factors are the LecA and LecB lectins. Both lectins play an important role in the adhesion to the host cells and display cytotoxic activity. In this study, we successfully synthesized hardly hydrolysable carbohydrate ligands targeting these pathogenic lectins, including two bispecific glycans. The interactions between LecA/LecB lectins and synthetic glycans were evaluated using hemagglutination (yeast agglutination) inhibition assays, comparing their efficacy with corresponding monosaccharides. Additionally, the binding affinities of bispecific glycans were assessed using isothermal titration calorimetry (ITC). Structural insight into the lectin-ligand interaction was obtained by determining the crystal structures of LecA/LecB lectins in complex with one of the bispecific ligands using X ray crystallography. This comprehensive investigation into the inhibitory potential of synthetic glycosides against P. aeruginosa lectins sheds light on their potential application in antimicrobial therapy.

Limited Evidence of Spillover of Antimicrobial-Resistant Klebsiella pneumoniae from Animal/Environmental Reservoirs to Humans in Vellore, India.

Klebsiella pneumoniae is a common opportunistic pathogen in humans, often associated with both virulence and antimicrobial resistance (AMR) phenotypes. K. pneumoniae have a highly plastic genome and can act as a vehicle for disseminating genetic information. Aiming to assess the impact of the human-animal-environment interface on AMR dissemination in K. pneumoniae we sampled and genome sequenced organisms from a range of environments and compared their genetic composition. Representative K. pneumoniae isolated from clinical specimens (n = 59), livestock samples (n = 71), and hospital sewage samples (n = 16) during a two-year surveillance study were subjected to whole genome sequencing. We compared the taxonomic and genomic distribution of K. pneumoniae, AMR gene abundance, virulence gene composition, and mobile genetic elements between the three sources. The K. pneumoniae isolates originating from livestock were clonally distinct from those derived from clinical/hospital effluent samples. Notably, the clinical and hospital sewage isolates typically possessed a greater number of resistance/virulence genes than those from animals. Overall, we observed a limited overlap of K. pneumoniae clones, AMR genes, virulence determinants, and plasmids between the different settings. In this setting, the spread of XDR and hypervirulent clones of K. pneumoniae appears to be restricted to humans with no obvious association with non-clinical sources. Emergent clones of K. pneumoniae carrying both resistance and virulence determinants are likely to have emerged in hospital settings rather than in animal or natural environments. These data challenge the current view of AMR transmission in K. pneumoniae in a One-Health context.

Psychrobacter sanguinis infection in a pediatric patient with craniopharyngioma identified in a blood culture by 16S rRNA sequencing

AbstractPsychrobacter species are gram‐negative bacteria in the Moraxellaceae family. These bacteria are considered rare opportunistic human pathogens, and the infection sites include blood, cerebral spinal fluid, wounds, urine, the ears, and the eyes. Few cases of human infection by these species have been described previously. We report a case of a 10‐year‐old boy with postneurosurgical bacteremia due to Psychrobacter sanguinis infection. This infection was difficult to identify using routine biochemical phenotypical tests. Sequencing of 16S rRNA was performed to identify this pathogen. The patient was successfully treated with antibiotics. In conclusion, P. sanguinis infections are rare but should be considered when cultures remain negative for common pathogens.

Acanthamoeba polyphaga de novo transcriptome and its dynamics during Mimivirus infection

Mimivirus bradfordmassiliense (Mimivirus) is a giant virus that infects Acanthamoeba species – opportunistic human pathogens. Long- and short-read sequencing were used to generate a de novo transcriptome of the host and followed the dynamics of both host and virus transcriptomes over the course of infection. The assembled transcriptome of the host included 22,604 transcripts and 13,043 genes, with N50 = 2,372 nucleotides. Functional enrichment analysis revealed major changes in the host transcriptome, namely, enrichment in downregulated genes associated with cytoskeleton homeostasis and DNA replication, repair, and nucleotide synthesis. These modulations, together with those implicated by other enriched processes, indicate cell cycle arrest, which was demonstrated experimentally. We also observed upregulation of host genes associated with transcription, secretory pathways and, as reported here for the first time, peroxisomes and the ubiquitin-proteasome system. In Mimivirus, the early stages of infection were marked by upregulated genes related to DNA replication, transcription, translation, and nucleotide metabolism, and in later stages, enrichment in genes associated with lipid metabolism, carbohydrates, and proteases. Some of the changes observed in the amoebal transcriptome likely point to Mimivirus infection causing dismantling of host cytoskeleton and translocation of endoplasmic reticulum membranes to viral factory areas.

Designing potential lead compounds targeting aminoglycoside N (6′)-acetyltransferase in Serratia marcescens: A drug discovery strategy

Serratia marcescens is an opportunistic human pathogen that causes urinary tract infections, ocular lens infections, and respiratory tract infections. S. marcescens employs various defense mechanisms to evade antibiotics, one of which is mediated by aminoglycoside N-acetyltransferase (AAC). In this mechanism, the enzyme AAC facilitates the transfer and linkage of the acetyl moiety from the donor substrate acetyl-coenzyme A to specific positions on antibiotics. This modification alters the antibiotic's structure, leading to the inactivation of aminoglycoside antibiotics. In the current scenario, antibiotic resistance has become a global threat, and targeting the enzymes that mediate resistance is considered crucial to combat this issue. The study aimed to address the increasing global threat of antibiotic resistance in Serratia marcescens by targeting the aminoglycoside N-acetyltransferase (AAC (6′)) enzyme, which inactivates aminoglycoside antibiotics through acetylation. Due to the absence of experimental structure, we constructed a homology model of aminoglycoside N (6′)-acetyltransferase (AAC (6′)) of S. marcescens using the atomic structure of aminoglycoside N-acetyltransferase AAC (6′)-Ib (PDB ID: 1V0C) as a template. The stable architecture and integrity of the modelled AAC (6′) structure were analyzed through a 100 ns simulation. Structure-guided high-throughput screening of four small molecule databases (Binding, Life Chemicals, Zinc, and Toslab) resulted in the identification of potent inhibitors against AAC (6′). The hits obtained from screening were manually clustered, and the five hit molecules were shortlisted based on the docking score, which are observed in the range of −17.09 kcal/mol to −11.95 kcal/mol. These selected five molecules displayed acceptable pharmacological properties in ADME predictions. The binding free energy calculations, and molecular dynamics simulations of ligand bound AAC (6′) complexes represented higher affinity and stable binding. The selected molecules demonstrated stable binding with AAC (6′), indicating their strong potential to hamper the binding of aminoglycoside in the respective site. and thereby inhibit. This process mitigates enzyme mediated AAC (6′) activity on aminoglycosides and reverse the bactericidal function of aminoglycosides, and also this method could serve as a platform for the development of potential antimicrobials.

PneumoBrowse 2: an integrated visual platform for curated genome annotation and multiomics data analysis of Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic human pathogen responsible for high morbidity and mortality rates. Extensive genome sequencing revealed its large pangenome, serotype diversity, and provided insight into genome dynamics. However, functional genome analysis has lagged behind, as that requires detailed and time-consuming manual curation of genome annotations and integration of genomic and phenotypic data. To remedy this, PneumoBrowse was presented in 2018, a user-friendly interactive online platform, which provided the detailed annotation of the S. pneumoniae D39V genome, alongside transcriptomic data. Since 2018, many new studies on S. pneumoniae genome biology and protein functioning have been performed. Here, we present PneumoBrowse 2 (https://veeninglab.com/pneumobrowse), fully rebuilt in JBrowse 2. We updated annotations for transcribed and transcriptional regulatory features in the D39V genome. We added genome-wide data tracks for high-resolution chromosome conformation capture (Hi-C) data, chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq), ribosome profiling, CRISPRi-seq gene essentiality data and more. Additionally, we included 18 phylogenetically diverse S. pneumoniae genomes and their annotations. By providing easy access to diverse high-quality genome annotations and links to other databases (including UniProt and AlphaFold), PneumoBrowse 2 will further accelerate research and development into preventive and treatment strategies, through increased understanding of the pneumococcal genome.

Complete genome sequence of Sphingobacterium multivorum strain FW102 with β-galactosidase activity in drinking fountain water.

Sphingobacterium multivorum is a ubiquitous environmental microbe in nature, particularly abundant in soil and water sources. Recently, S. multivorum has been considered to be a potential opportunistic pathogen in humans. We report the 5,9540,064-bp genome sequence of S. multivorum strain FW102, isolated from drinking fountain water and predicted to have multiple copies of β-galactosidase genes.

Development of two quantitative PCR assays for the detection of emerging opportunistic human pathogens belonging to the genus Phytobacter in routine diagnostics

The genus Phytobacter (fam. Enterobacteriaceae) includes species like Phytobacter diazotrophicus and Phytobacter ursingii, which have emerged as opportunistic human pathogens, particularly affecting vulnerable populations such as pre-term infants and immunocompromised patients. Traditional biochemical and molecular methods have struggled to accurately identify Phytobacter species in clinical diagnostics. This study addresses the issue by developing and validating two quantitative PCR (qPCR) assays using SYBR® Green I and TaqMan® technologies, targeting the nitrogen fixation regulatory gene (nifL) of Phytobacter spp. The SYBR® Green I assay showed a detection limit of a single cell per reaction, while the TaqMan® assay was easier to interpret due to the absence of background noise. These assays, validated with clinical isolates from Brazil, identified multiple new Phytobacter isolates, including a potentially novel species, providing improved diagnostic tools for detecting Phytobacter spp. and aiding in better clinical management.

CodY controls the SaeR/S two-component system by modulating branched-chain fatty acid synthesis in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive, opportunistic human pathogen that is a leading cause of skin and soft tissue infections and invasive disease worldwide. Virulence in this bacterium is tightly controlled by a network of regulatory factors. One such factor is the global regulatory protein CodY. CodY links branched-chain amino acid sufficiency to the production of surface-associated and secreted factors that facilitate immune evasion and subversion. Our previous work revealed that CodY regulates virulence factor gene expression indirectly in part by controlling the activity of the SaeRS two-component system (TCS). While this is correlated with an increase in membrane anteiso-15:0 and -17:0 branched-chain fatty acids (BCFAs) derived from isoleucine, the true mechanism of control has remained elusive. Herein, we report that CodY-dependent regulation of SaeS sensor kinase activity requires BCFA synthesis. During periods of nutrient sufficiency, BCFA synthesis and Sae TCS activity are kept relatively low by CodY-dependent repression of the ilv-leu operon and the isoleucine-specific permease gene brnQ2. In a codY null mutant, which simulates extreme nutrient limitation, de-repression of ilv-leu and brnQ2 directs the synthesis of enzymes in redundant de novo and import pathways to upregulate production of BCFA precursors. Overexpression of brnQ2, independent of CodY, is sufficient to increase membrane anteiso BCFAs, Sae-dependent promoter activity, and SaeR ~P levels. Our results further clarify the molecular mechanisms by which CodY controls virulence in S. aureus.IMPORTANCEExpression of bacterial virulence genes often correlates with the exhaustion of nutrients, but how the signaling of nutrient availability and the resulting physiological responses are coordinated is unclear. In S. aureus, CodY controls the activity of two major regulators of virulence-the Agr and Sae two-component systems (TCSs)-by unknown mechanisms. This work identifies a mechanism by which CodY controls the activity of the sensor kinase SaeS by modulating the levels of anteiso branched-chain amino acids that are incorporated into the membrane. Understanding the mechanism adds to our understanding of how bacterial physiology and metabolism are linked to virulence and underscores the role virulence in maintaining homeostasis. Understanding the mechanism also opens potential avenues for targeted therapeutic strategies against S. aureus infections.

Analysis of Antibiotic Response in Clinical Wound Pseudomonas aeruginosa Isolates: Unveiling Proteome Dynamics of Tobramycin-Tolerant Phenotype

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen, causing serious chronic infections. P. aeruginosa can adapt efficiently to antibiotic stressors via different genotypic or phenotypic strategies such as resistance and tolerance. The adaptation regulatory system is not always very well understood. In this study, we use shotgun proteomics to investigate the system-level response to tobramycin in two clinical wound P. aeruginosa isolates and PAO1. We profiled each strain for its antibiotic drug-tolerant phenotype using supra-minimum inhibitory concentrations (supra-MIC) of tobramycin and applied proteomics to investigate the protein expression profiles. The MIC revealed that all isolates were susceptible to tobramycin but at supra-MIC concentrations at stationary growth, a degree of tolerance was observed for the isolates. We identified around 40 % of the total proteins encoded by the P. aeruginosa genome and highlighted shared and unique protein signatures for all isolates. Comparative proteome profiling in the absence of antibiotic treatment showed divergent fingerprints, despite similarities in the growth behavior of the isolates. In the presence of tobramycin, the isolates shared a common response in the downregulation of proteins involved in the two-component system, whereas stress response proteins were present at higher levels. Our findings provide insight into the use of proteomic tools to dissect the system-level response in clinical isolates in the absence and presence of antibiotic stress.

Interactions between Pseudomonas aeruginosa and six opportunistic pathogens cover a broad spectrum from mutualism to antagonism.

Bacterial infections often involve more than one pathogen. While it is well established that polymicrobial infections can impact disease outcomes, we know little about how pathogens interact and affect each other's behaviour and fitness. Here, we used a microscopy approach to explore interactions between Pseudomonas aeruginosa and six human opportunistic pathogens that often co-occur in polymicrobial infections: Acinetobacter baumannii, Burkholderia cenocepacia, Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, and Staphylococcus aureus. When following growing microcolonies on agarose pads over time, we observed a broad spectrum of species-specific ecological interactions, ranging from mutualism to antagonism. For example, P. aeruginosa engaged in a mutually beneficial interaction with E. faecium but suffered from antagonism by E. coli. While we found little evidence for active directional growth towards or away from cohabitants, we observed that some pathogens increased growth in double layers in response to competition and that physical forces due to fast colony expansion had a major impact on fitness. Overall, our work provides an atlas of pathogen interactions, highlighting the diversity of potential species dynamics that may occur in polymicrobial infections. We discuss possible mechanisms driving pathogen interactions and offer predictions of how the different ecological interactions could affect virulence.

Study on Pseudomonas spp. isolated from pork and chicken meat

Pseudomonas spp. are heterotrophic, Gram-negative, aerobic bacteria widely distributed in the environment, acting as food spoilage agents and opportunistic pathogens in humans. In this study, we applied the ISO 13720:2010 for enumeration of presumptive Pseudomonas spp. in meat and meat products to investigate the contamination of Pseudomonas spp. in pork and chicken meat samples collected from markets in various districts of Ho Chi Minh City. The results indicated that 78% of pork samples and 72% of chicken samples were contaminated with Pseudomonas spp., with average densities of 4.9×106 CFU/g and 6.8×106 CFU/g, respectively. Almost isolated Pseudomonas strains could grow at neutral and alkaline pH, with an optimal temperature range of 10°C to 44°C, and tolerate to NaCl 5%. These findings provide critical information on the distribution and growth characteristics of Pseudomonas spp. in meat, therefore, being a premise to propose the research on density thresholds to ensure food quality.

Transition metal homoeostasis is key to metabolism and drug tolerance of Mycobacterium abscessus

AbstractAntimicrobial resistance (AMR) is one of the major challenges humans are facing this century. Understanding the mechanisms behind the rise of AMR is therefore crucial to tackling this global threat. The presence of transition metals is one of the growth-limiting factors for both environmental and pathogenic bacteria, and the mechanisms that bacteria use to adapt to and survive under transition metal toxicity resemble those correlated with the rise of AMR. A deeper understanding of transition metal toxicity and its potential as an antimicrobial agent will expand our knowledge of AMR and assist the development of therapeutic strategies. In this study, we investigate the antimicrobial effect of two transition metal ions, namely cobalt (Co2+) and nickel (Ni2+), on the non-tuberculous environmental mycobacterium and the opportunistic human pathogen Mycobacterium abscessus. The minimum inhibitory concentrations of Co2+ and Ni2+ on M. abscessus were first quantified and their impact on the bacterial intracellular metallome was investigated. A multi-omics strategy that combines transcriptomics, bioenergetics, metabolomics, and phenotypic assays was designed to further investigate the mechanisms behind the effects of transition metals. We show that transition metals induced growth defect and changes in transcriptome and carbon metabolism in M. abscessus, while the induction of the glyoxylate shunt and the WhiB7 regulon in response to metal stresses could be the key response that led to higher AMR levels. Meanwhile, transition metal treatment alters the bacterial response to clinically relevant antibiotics and enhances the uptake of clarithromycin into bacterial cells, leading to increased efficacy. This work provides insights into the tolerance mechanisms of M. abscessus to transition metal toxicity and demonstrates the possibility of using transition metals to adjuvant the efficacy of currently using antimicrobials against M. abscessus infections.

Complex trauma sequelae: Mycobacterium goodii and Priestia endophytica Hardware infection in a patient with Ehlers-Danlos syndrome

A 26-year-old man with Ehlers-Danlos syndrome, recurrent otitis externa, and chronic otitis media sustained a left lower extremity amputation and open femur fracture with internal hardware fixation after a motor vehicle collision in Arizona. He presented to the emergency department at our institution with severe left leg pain and purulent discharge despite receiving two unidentified antibiotics upon discharge. Evaluations revealed an abscess and malunion of the femur. Initial cultures yielded scant Priestia endophytica, leading to daptomycin treatment. His condition worsened until Gram-positive bacilli identified as Mycobacterium goodii, a rare nosocomial mycobacterial species, were found. Significant improvement occurred with appropriate antibiotics. This case highlights the challenges in diagnosing and managing M. goodii infections in immunocompromised patients with orthopedic complications and notes P. endophytica as a previously unreported, possibly opportunistic human pathogen.

Micronutrient availability alters Candida albicans growth and farnesol accumulation: implications for studies using RPMI-1640.

The dimorphic fungus Candida albicans is a major opportunistic pathogen of humans. RPMI-1640 is a chemically defined growth medium commonly used with C. albicans. We identified over 32,000 publications with keywords RPMI and C. albicans. Additionally, Antifungal Susceptibility Testing (AFST) protocols in the United States (CLSI) and Europe (EUCAST) utilize RPMI as a base media to assess drug efficacy against clinical fungal isolates. RPMI contains many nutrients but no added trace metals. We found that the growth characteristics with RPMI were dependent on which MOPS buffer was chosen and the contamination of that buffer by trace levels of Mn(II) and Zn(II). Added Mn(II) was most needed for cell growth while added Zn(II) was most needed for secretion of farnesol and other signaling molecules.

Diverse signatures of convergent evolution in cactus-associated yeasts.

Many distantly related organisms have convergently evolved traits and lifestyles that enable them to live in similar ecological environments. However, the extent of phenotypic convergence evolving through the same or distinct genetic trajectories remains an open question. Here, we leverage a comprehensive dataset of genomic and phenotypic data from 1,049 yeast species in the subphylum Saccharomycotina (Kingdom Fungi, Phylum Ascomycota) to explore signatures of convergent evolution in cactophilic yeasts, ecological specialists associated with cacti. We inferred that the ecological association of yeasts with cacti arose independently approximately 17 times. Using a machine learning-based approach, we further found that cactophily can be predicted with 76% accuracy from both functional genomic and phenotypic data. The most informative feature for predicting cactophily was thermotolerance, which we found to be likely associated with altered evolutionary rates of genes impacting the cell envelope in several cactophilic lineages. We also identified horizontal gene transfer and duplication events of plant cell wall-degrading enzymes in distantly related cactophilic clades, suggesting that putatively adaptive traits evolved independently through disparate molecular mechanisms. Notably, we found that multiple cactophilic species and their close relatives have been reported as emerging human opportunistic pathogens, suggesting that the cactophilic lifestyle-and perhaps more generally lifestyles favoring thermotolerance-might preadapt yeasts to cause human disease. This work underscores the potential of a multifaceted approach involving high-throughput genomic and phenotypic data to shed light onto ecological adaptation and highlights how convergent evolution to wild environments could facilitate the transition to human pathogenicity.

Discovery of a Small-Molecule Inhibitor Targeting the Biofilm Regulator BrpT in Vibrio vulnificus.

Vibrio vulnificus, an opportunistic human pathogen, employs biofilm formation as a key survival and virulence mechanism. BrpT, a transcriptional regulator, is essential for V. vulnificus biofilm development by regulating the expression of biofilm-related genes. In this study, we aimed to identify a small molecule inhibitor of BrpT to combat V. vulnificus biofilm formation. High-throughput screening of 7,251 compounds using an Escherichia coli reporter strain carrying the arabinose-inducible brpT gene and a BrpT-activated promoter fused to the luxCDABE operon identified a hit compound, BTI (BrpT Inhibitor). BTI potently inhibited BrpT activity in V. vulnificus (EC50 of 6.48 μM) without affecting bacterial growth or host cell viability. Treatment with BTI significantly reduced the expression of the BrpT regulon and impaired biofilm formation and colony rugosity in V. vulnificus, thus increasing its susceptibility to antibiotics. In vitro biochemical analyses revealed that BTI directly binds to BrpT and inhibits its transcriptional regulatory activity. The identification of BTI as a specific inhibitor of BrpT that effectively diminishes V. vulnificus biofilm formation provides a promising foundation for the development of novel anti-biofilm strategies, with the potential to address the growing challenge of antibiotic resistance and improve the treatment of biofilm-associated infections.

DivIVA controls the dynamics of septum splitting and cell elongation in Streptococcus pneumoniae.

Bacterial shape and division rely on the dynamics of cell wall assembly, which involves regulated synthesis and cleavage of the peptidoglycan. In ovococci, these processes are coordinated within an annular mid-cell region with nanometric dimensions. More precisely, the cross-wall synthesized by the divisome is split to generate a lateral wall, whose expansion is insured by the insertion of the so-called peripheral peptidoglycan by the elongasome. Septum cleavage and peripheral peptidoglycan synthesis are, thus, crucial remodeling events for ovococcal cell division and elongation. The structural DivIVA protein has long been known as a major regulator of these processes, but its mode of action remains unknown. Here, we integrate click chemistry-based peptidoglycan labeling, direct stochastic optical reconstruction microscopy, and in silico modeling, as well as epifluorescence and stimulated emission depletion microscopy to investigate the role of DivIVA in Streptococcus pneumoniae cell morphogenesis. Our work reveals two distinct phases of peptidoglycan remodeling during the cell cycle that are differentially controlled by DivIVA. In particular, we show that DivIVA ensures homogeneous septum cleavage and peripheral peptidoglycan synthesis around the division site and their maintenance throughout the cell cycle. Our data additionally suggest that DivIVA impacts the contribution of the elongasome and class A penicillin-binding proteins to cell elongation. We also report the position of DivIVA on either side of the septum, consistent with its known affinity for negatively curved membranes. Finally, we take the opportunity provided by these new observations to propose hypotheses for the mechanism of action of this key morphogenetic protein.IMPORTANCEThis study sheds light on fundamental processes governing bacterial growth and division, using integrated click chemistry, advanced microscopy, and computational modeling approaches. It addresses cell wall synthesis mechanisms in the opportunistic human pathogen Streptococcus pneumoniae, responsible for a range of illnesses (otitis, pneumonia, meningitis, septicemia) and for one million deaths every year worldwide. This bacterium belongs to the morphological group of ovococci, which includes many streptococcal and enterococcal pathogens. In this study, we have dissected the function of DivIVA, which is a structural protein involved in cell division, morphogenesis, and chromosome partitioning in Gram-positive bacteria. This work unveils the role of DivIVA in the orchestration of cell division and elongation along the pneumococcal cell cycle. It not only enhances our understanding of how ovoid bacteria proliferate but also offers the opportunity to consider how DivIVA might serve as a scaffold and sensor for particular membrane regions, thereby participating in various cell cycle processes.