Lignin is a vast yet underutilized source of renewable energy. The microbial valorization of lignin is challenging due to the toxicity of its degradation intermediates, particularly formaldehyde. In this study, we engineered Methylobacterium extorquens PA1 to metabolize lignin-derived methoxylated aromatics, vanillate (VA) and protocatechuate (PCA), by introducing the van and pca gene clusters. Compared to Pseudomonas putida, M. extorquens PA1 exhibited better formaldehyde detoxification, enabling robust growth on VA without accumulation of formaldehyde. Genetic analyses confirmed that formaldehyde oxidation and stress response systems, rather than C1 assimilation, were important for VA metabolism. Additionally, VA and PCA were found to disrupt membrane potential, contributing to their inherent toxicity. Our findings establish M. extorquens PA1 as a promising chassis for lignin valorization and provide a framework for engineering formaldehyde-resistant microbial platforms.IMPORTANCEIn developing biotechnological solutions for a circular economy, it is critical to valorize all parts of renewable resources, such as lignocellulose from vegetative components of agricultural crops and from bioenergy feedstocks. After chemical breakdown, the aromatics arising from lignin present significant challenges for use due to their toxicity. Here, we address one component of this challenge-the methoxy groups that get released as formaldehyde-and show that existing biotechnological platform organisms with strong formaldehyde metabolism, such as Methylobacterium extorquens, can be transformed into highly capable utilizers of methoxylated aromatics.

All biological products are required to demonstrate the absence of adventitious viruses (AVs), which may be inadvertently introduced at different steps involved in the manufacturing process. The currently recommended in vitro and in vivo virus detection assays have limitations for broad detection and are lengthy and laborious. Additionally, the use of animals is discouraged by the global 3 R's initiative for replacement, reduction, and refinement. High-throughput or next-generation sequencing (HTS/NGS) technologies can rapidly detect known and novel viruses in biological materials. There are, however, challenges for HTS detection of AVs due to differential abundance of viral sequences in public databases, which led to the creation of a non-redundant, Reference Viral Database (RVDB) containing all viral, viral-like, and viral-related sequences, with a reduced cellular sequence content. In this paper, we describe improvements in RVDB, which include the transition of RVDB production scripts from the original Python 2 to Python 3 codebase, updating the semantic pipeline to remove misannotated non-viral sequences and irrelevant viral sequences, use of taxonomy for the removal of phages, and inclusion of a quality-check step for SARS-CoV-2 genomes to exclude low-quality sequences. Additionally, RVDB website updates include search tools for exploring the database sequences and implementation of an automatic pipeline for providing annotation information to distinguish non-viral and viral sequences in the database. These updates for refining RVDB are expected to enhance HTS bioinformatics by reducing the computational time and increasing the accuracy for virus detection.IMPORTANCEHigh-throughput sequencing (HTS) has emerged as an advanced technology for demonstrating the safety of biological products. HTS can be used as an alternative adventitious virus detection method for replacing the currently recommended in vivo and PCR assays and supplementing or replacing the in vitro cell culture assays. However, HTS bioinformatics analysis for broad virus detection, including both known and novel viruses, depends on using a comprehensive and accurately annotated database. In this study, we have refined our original comprehensive Reference Virus Database (RVDB) for greater accuracy of virus detection with a reduced computational burden. Additionally, the production script for automating the generation of RVDB was updated to facilitate reliable database production and timely availability.

Antibiotic-resistant infections pose a growing threat to global health. This study reveals how genes conferring antibiotic resistance can move between bacteria that belong to different phyla lineages previously thought to be too evolutionarily distant for frequent gene exchange. By analyzing nearly 1 million resistance genes from over 400,000 bacterial genomes, the researchers uncovered hundreds of inter-phylum transfer events, exposing surprising patterns in how different classes of resistance genes spread. The findings highlight that conjugative systems are less common than expected in cross-phyla transfers and suggest that alternative mechanisms may play key roles. This new understanding of how resistance genes leap between vastly different bacterial groups can inform strategies to slow the emergence of drug-resistant infections, aiding in the development of more effective public health interventions.

Shigella is a leading cause of diarrheal morbidity and mortality in young children from low- and middle-income countries. Here, we aimed to verify the ability of the generalized modules for membrane antigens (GMMA)-based Shigella sonnei candidate vaccine 1790GAHB to elicit an anti-protein antibody response. Serum samples from previous clinical trials in adults (a dose-escalation study and its extension in France, a vaccine efficacy study after human challenge in the United States, and a study in Kenya) were investigated using pan-proteome microarrays consisting of 3,150 full-length or fragmented Shigella proteins. Pre-/post-vaccination comparisons identified subsets of proteins that were highly immunoreactive and largely overlapped across all trials; the T3SS lipochaperone family protein (expressed on GMMA) was the most reactive in all studies. Responses to several microarray antigens correlated well with S. sonnei LPS serum IgG antibody levels. Overall, we confirmed the ability of GMMA to elicit an anti-protein IgG/IgA response; however, no association with protection against shigellosis was identified. In the challenge study, IgG response to seven antigens (IpaC, IpaB, IpaA, IpaD, IpaH, IpgC, and MxiD; not expressed on GMMA) was associated with a decreased risk of shigellosis. These antigens were observed to also have high IgG responses at baseline in individuals naturally exposed to Shigella and could constitute targets for future vaccine development.IMPORTANCEShigella remains a major cause of diarrheal disease, especially in children aged under 5 years from low-to-middle-income countries. No vaccine against shigellosis is yet widely available despite the high public health need. An ideal vaccine would provide protection against the most prevalent species, Shigella flexneri and Shigella sonnei; therefore, it could be relevant to identify common antigens. We developed a microarray containing 3,150 full-length or fragmented proteins selected across Shigella species. Sera collected in four clinical trials conducted in three countries of varying endemicity to evaluate a S. sonnei GMMA-based candidate vaccine were tested against these proteins. We identified several Shigella proteins (IpaC, IpaB, IpaA, IpaD, IpaH, IpgC, MxiD) that induced robust antibody response following experimental challenge or natural infection. These proteins correlated with a reduced risk of shigellosis after the S. sonnei challenge. We found no apparent role for anti-GMMA proteins' IgG or IgA response in protection against shigellosis.

Klebsiella pneumoniae is a globally recognized microbial pathogen with significant clinical impact. The bacterium harbors the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which provide adaptive immunity against invading foreign nucleic acids. Recent studies suggest that certain CRISPR-Cas systems can regulate endogenous genes, influencing bacterial virulence. However, their role in regulating pathogenicity in K. pneumoniae remains poorly understood. This study investigates the regulatory role of the type I-E* CRISPR-Cas system in a hypervirulent K. pneumoniae strain, focusing on its impact on histidine metabolism and pathogenicity. Transcriptome analyses identified differentially expressed genes (DEGs) between the casABECD-deletion and wild-type strains, including significant upregulation of the histidine utilization (Hut) operon and downregulation of biofilm-related genes. These molecular changes resulted in enhanced histidine metabolic activity, reduced biofilm formation, attenuated virulence in A549 lung epithelial cells, and improved survival of Galleria mellonella, as validated through phenotypic and virulence assays. Our bioinformatic analysis indicated that the CRISPR-Cas system in K. pneumoniae targets the hutT sequence, which is part of the Hut operon. Furthermore, the overexpression of hutT mitigated CRISPR-Cas-mediated repression of the Hut operon, as observed in virulence assays, while simultaneous deletion of hutH and casABECD restored the reduced virulence in the ΔcasABECD strain. Additionally, deletion of casABECD significantly enhances the growth of the strain in medium with histidine as the sole carbon source, highlighting the intricate regulatory role of the CRISPR-Cas system in metabolic adaptation. Collectively, these findings uncover a novel role for the CRISPR-Cas system in regulating metabolic pathways and virulence in hypervirulent K. pneumoniae.IMPORTANCEClustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are primarily recognized for their roles in adaptive immunity against foreign genetic elements in bacteria. However, emerging evidence indicates that these systems can also regulate endogenous genes, thereby influencing bacterial physiology and virulence. In this study, we demonstrate that the type I-E* CRISPR-Cas system in Klebsiella pneumoniae targets the hutT gene, a critical component of the histidine utilization (Hut) pathway. This targeting potentially impacts hutT transcription and alters the expression of other hut genes, ultimately enhancing bacterial virulence. Our findings reveal a previously unrecognized regulatory mechanism through which CRISPR-Cas systems facilitate metabolic adaptation and pathogenicity in K. pneumoniae. This study broadens our understanding of the multifaceted roles of CRISPR-Cas systems in bacterial physiology and pathobiology, with implications for clinically relevant pathogens.

Putrescine is an important interspecies and interkingdom communication signal, modulating the bacterial motility, biofilm formation, and virulence of D. oryzae. The understanding of the regulation of putrescine biosynthesis and transport in D. oryzae is limited. In this study, we report that alarmone ppGpp hierarchically modulates putrescine biosynthesis and transport and synergistically cooperates with putrescine to regulate virulence traits and the virulence of D. oryzae. We found that the alarmone ppGpp synthesized by RelA regulated putrescine biosynthesis through modulating speA expression, and the product putrescine would thus inhibit the expression of potF and plaP. Remarkably, we unveiled the synergistic effect of alarmone ppGpp and putrescine on the modulation of swimming motility and zeamine production. Compared with the single deletion of either relA or speA, the double deletion of relA and speA could decrease the expression of RNA chaperone encoded gene hfq and the production of phytotoxin zeamine, which further attenuated the capability of D. oryzae EC1 in inhibition of rice seed germination. Collectively, the findings from this study depict alarmone ppGpp regulation on putrescine biosynthesis and transport and present the cooperation of regulation of alarmone ppGpp and putrescine in the virulence of D. oryzae.IMPORTANCEDickeya oryzae is the causal agent of rice root rot disease. Bacterial motility and phytotoxic zeamines are characterized as two major virulent factors during D. oryzea infecting rice seed. Putrescine, as an interspecies and interkingdom communication signal for the infections of D. oryzae, has been previously demonstrated to be involved in the modulation of bacterial motility. Here we report the novel synergistic effect of putrescine signal and alarmone ppGpp on the regulation of both zeamine production and bacterial motility via modulating the expression of RNA chaperone-encoded gene hfq. In addition, we also showed that alarmone ppGpp hierarchically modulates putrescine biosynthesis and transport. Therefore, the findings of this study unveil the previously undetermined contribution of putrescine in the modulation of virulence determinants, and the regulatory mechanism of putrescine biosynthesis and transport in D. oryzae.

Persistent bacterial airway infection is a hallmark feature of cystic fibrosis (CF). Achromobacter spp. are gram-negative rods that can cause persistent airway infection in people with CF (pwCF), but the knowledge of host immune responses to these bacteria is limited. The aim of this study was to investigate if patients develop antibodies against Achromobacter xylosoxidans, the most common Achromobacter species, and to identify the bacterial antigens that induce specific IgG responses. Seven serum samples from pwCF with Achromobacter infection were screened for antibodies against bacteria in an ELISA coated with A. xylosoxidans, A. insuavis, or Pseudomonas aeruginosa. Sera from pwCF with or without P. aeruginosa infection (n = 22 and 20, respectively) and healthy donors (n = 4) were included for comparison. Serum with high titers to A. xylosoxidans was selected for affinity purification of bacterial antigens using serum IgGs bound to protein G beads. The resulting IgG-antigen complexes were then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Selected antigens of interest were produced in recombinant form and used in an ELISA to confirm the results. Four of the seven patients with Achromobacter infection had serum antibodies against Achromobacter. Using patient serum-IgG for affinity purification of A. xylosoxidans proteins, we identified eight antigens. Three of these, which were not targeted by anti-P. aeruginosa antibodies, were expressed recombinantly for further validation: dihydrolipoyl dehydrogenase (DLD), type I secretion C-terminal target domain-containing protein, and domain of uncharacterized function 336 (DUF336). While specific IgG against all three recombinant antigens was confirmed in the patient serum with high titers against Achromobacter, DLD and DUF336 showed the least binding to serum IgG from pwCF without Achromobacter spp. infection. Using serum IgG affinity purification in combination with LC-MS/MS and confirming the results using ELISA against recombinant proteins, we have identified bacterial antigens from A. xylosoxidans.IMPORTANCEAchromobacter species are opportunistic pathogens that can cause airway infections in people with cystic fibrosis. In this patient population, persistent Achromobacter infection is associated with low lung function, but the knowledge about bacterial interactions with the host is currently limited. In this study, we identify protein antigens that induce specific antibody responses in the host. The identified antigens may potentially be useful in serological assays, serving as a complement to culturing methods for the diagnosis and surveillance of Achromobacter infection.

Infectious diseases remain a major cause of global mortality, yet basic questions concerning the relationship between within-host processes governing pathogen burden (pathogen replication, immune responses) and population-scale (epidemiological) patterns of mortality remain obscure. We use a structured literature review to leverage the extensive biomedical data generated by controlled host infections to address the epidemiological question of whether infection-induced mortality is constant, accelerating, or follows some other pattern of change and to infer the within-host mechanistic basis of this pattern. We show that across diverse lethal infection models, the risk of death increases approximately exponentially in time since infection, in a manner phenomenologically similar to the dynamics of all-cause death. We further show that this pattern of accelerating risk is consistent with multiple alternate mechanisms of pathogen growth and host-pathogen interaction, underlining the limitations of current experimental approaches to connect within-host processes to epidemiological patterns. We review critical experimental questions that our work highlights, requiring additional non-invasive data on pathogen burden throughout the course of infection.IMPORTANCEHere, we ask a simple question: what are the dynamics of pathogen-induced death? Death is a central phenotype in both biomedical and epidemiological infectious disease biology, yet very little work has attempted to link the biomedical focus on pathogen dynamics within a host and the epidemiological focus on populations of infected hosts. To systematically characterize the dynamics of death in controlled animal infections, we analyzed 209 data sets spanning diverse lethal animal infection models. Across experimental models, we find robust support for an accelerating risk of death since the time of infection, contrasting with conventional epidemiological models that assume a constant elevated risk of death. Using math models, we show that multiple processes of growth and virulence are consistent with accelerating risk of death, and we end with a discussion of critical experiments to resolve how within-host biomedical processes map onto epidemiological patterns of disease.

The world has been facing an upsurge in cholera cases since 2021, a similar trend continuing into 2022, with over 29 countries reporting cholera outbreaks (World Health Organization, 16 December 2022, Disease Outbreak News, Cholera-global situation). Treatment of cholera involves oral rehydration therapy coupled with antibiotics to reduce the duration of the illness. However, in recent years, indiscriminate use of antibiotics has contributed to the emergence of antibiotic-resistant strains. In this study, we have addressed the problem of antibiotic resistance by targeting virulence factors. Screening various compounds using in silico methods led to the identification of a small molecule, SB, that inhibits the virulence cascade in V. cholerae. We demonstrated that (i) SB intervened in ToxT protein-DNA binding and subsequently affected the expression of ToxT-regulated virulence genes (ctxAB and tcpA) and (ii) SB is a potential therapeutic candidate for the development of a novel antimicrobial agent.

The Ganges delta of the Bay of Bengal is a recognized hotspot for the emergence and spread of novel variants of Vibrio cholerae. Despite being a diverse species, very little information is available concerning environmental and human-associated aspects of V. cholerae serogroups, other than the two major epidemic-related serogroups O1 and O139. This represents a crucial gap in understanding the spectrum of diversity, ecology, and epidemiology of the species influencing the dynamics of global cholera. In this study, we describe an emerging variant of V. cholerae displaying the antigenic property of serogroup O47, associated with a cholera-like outbreak in coastal Bangladesh where cholera has been endemic for centuries. This outbreak coincides with a rise in cases of cholera caused by V. cholerae O1, as well as frequency of isolation of serogroups O47 and O1 from the environment. The V. cholerae O47 isolates proved clonal in nature, and their genome biology revealed distinct features, with respect to multidrug resistance (MDR), serogroup-specific genes, genomic island combinations, and overall phylogenetic properties. Genome comparison confirmed the absence of canonical virulence factors of V. cholerae O1 and O139, namely, cholera toxin (CTX) and toxin-co-regulated pili (TCP), and the presence of putative virulence factors including type 3 secretion system (T3SS) and an MDR pseudo-compound transposon, carrying genes for macrolide resistance and extended spectrum beta-lactamase. Results of the study suggest that V. cholerae O47 could represent an emerging Vibrio pathogen with the potential to spread virulence and antimicrobial resistance traits impacting the management of cholera-like diseases.IMPORTANCEDespite the global insurgence of human diseases caused by Vibrios in recent years, most research focuses only on the O1 serogroup of V. cholerae, leaving a significant gap concerning the environmental and human-associated aspects of other serogroups found in nature. Although other serogroups are often found associated with sporadic diarrhea cases, in 1992-1993, a massive cholera-like diarrhea epidemic was initiated by a "non-O1" serogroup, namely, O139 that temporally displaced O1 from endemic cholera in the Bay of Bengal villages of Bangladesh and India, highlighting the potential threat they might pose. This study describes yet another emerging variant of V. cholerae, displaying the antigenic property of serogroup O47, associated with a cholera-like outbreak in a coastal locality in Bangladesh. Findings of the study offer critical insights into the genome biology of V. cholerae O47 and its potential implications for understanding their ecology and epidemiology of cholera-like diseases.