Biology subjects

Severin, G. B.

Publications and source records attributed to Severin, G. B..

7 recordsLinked to original sources

bioRxiv2024

Multiplexed PCR to measure in situ growth rates of uropathogenic E. coli during experimental urinary tract infection.

Measuring bacterial growth rates in vitro is routine, however, determining growth rates during infection within a host has been more challenging. Peak-to-trough ratio (PTR) is a technique for studying microbial growth dynamics, calculated using the ratio of replication origin (ori) copies to those of the terminus (ter), as originally defined by whole genome sequencing (WGS). WGS presents significant challenges in terms of expense and data analysis complexity due to the presence of host DNA in the samples. Here, we used multiplexed PCR with fluorescent probes to estimate bacterial growth rates based on the abundance of ori- and ter-adjacent loci, without the need for WGS. We establish the utility of this approach by comparing growth rates of the uropathogenic Escherichia coli (UPEC) strain HM86 by WGS (PTR) and qPCR to measure the equivalent ori:ter (O:TPCR). We found that PTR and O:TPCR were highly correlated and that O:TPCR reliably predicted growth rates calculated by conventional methods. O:TPCR was then used to calculate the in situ E. coli growth rates in urine, bladder, and kidneys collected over the course of a week from a murine model of urinary tract infection (UTI). These analyses revealed that growth rate of UPEC strains gradually increased during the early stages of infection (0-6h), followed by a slow decrease in growth rates during later time points (1-7 days). This rapid and convenient method provides valuable insights into bacterial growth dynamics during infection and can be applied to other bacterial species in both animal models and clinical infections. ImportanceAccurately measuring bacterial growth rates in the host, which plays a crucial role in determining the success of pathogens in establishing infections, poses significant challenges. To address this, bacterial replication rate has been measured as a proxy for growth rate estimation. While whole genome sequencing (WGS) has been used for this purpose, it comes with drawbacks such as high costs and difficulties in analyzing bacterial sequences due to the overwhelming presence of host DNA. In this study, we validate a more accessible PCR-based approach compared to the established WGS method and confirmed the reliability of our PCR-based technique. We then applied it to measure the growth rate of Escherichia coli during experimental urinary tract infection in a mouse model. This study provides a cost-effective and efficient alternative to WGS for studying bacterial replication dynamics during infection, potentially offering new insights into pathogen behavior and host-microbe interactions.

microbiology↗

bioRxiv2024

Negative feedback of cyclic di-GMP levels optimizes switching between sessile and motile lifestyles in Vibrio cholerae

The signaling molecule cyclic di-GMP (c-di-GMP) controls the switch between bacterial motility and biofilm production, and fluctuations in cellular levels of c-di-GMP have been implicated in Vibrio cholerae pathogenesis. Intracellular concentrations of c-di-GMP are controlled by the interplay of diguanylate cyclase (DGC) enzymes, which synthesize c-di-GMP to promote biofilms, and phosphodiesterase (PDE) enzymes, which hydrolyze c-di-GMP to drive motility. To track the complete regulatory logic of how V. cholerae responds to changing c-di-GMP levels, we followed a time course of overexpression of either the V. campbellii diguanylate cyclase QrgB or a variant of QrgB lacking catalytic activity (QrgB*). We find that QrgB increases c-di-GMP levels relative to QrgB* for 30 minutes after overexpression, but the effect of QrgB on c-di-GMP levels plateaus at 30 minutes, indicating tight adaptive control of c-di-GMP levels. In contrast, loss of VpsR, a master regulator activating biofilm formation upon binding to c-di-GMP, leads to higher baseline levels of c-di-GMP and continuously increasing c-di-GMP through 60 minutes after QrgB induction, revealing the existence of a negative feedback loop on c-di-GMP levels operating through VpsR. Through a combination of RNA polymerase ChIP-seq, RNA-seq, and genetic approaches, we show that transcription of a gene encoding a PDE, cdgC, is activated by VpsR at high c-di-GMP concentrations, mediating this negative feedback on c-di-GMP levels. Further, although cells lacking cdgC exhibit enhanced biofilm formation, these mutants are outcompeted by wild type V. cholerae in colonization assays that reward a combination of attachment, dispersal, and motility behaviors. These results underscore the importance of negative feedback regulation of c-di-GMP to maintain appropriate homeostatic levels for efficient transitioning between biofilm formation and motility, both of which are necessary over the course of the V. cholerae infection cycle.

microbiology↗

bioRxiv2024

A novel method for integrating genomic and Tn-Seq data to identify common in vivo fitness mechanisms across multiple bacterial species

Sepsis is life-threatening organ dysfunction due to an unregulated immune response to infection. Bacteremia is a leading cause of sepsis, and members of the Enterobacterales cause nearly half of bacteremia cases annually. While previous Tn-Seq studies to identify novel bacteremia-fitness genes have provided valuable insight into virulence mechanisms, evidence for common pathways across species is lacking. To identify common fitness pathways in five bacteremia- caused Enterobacterales species, we utilized the JCVI pan-genome pipeline to integrate Tn-Seq fitness data with multiple available functional data types. Core genes from species pan-genomes were used to construct a multi-species core pan-genome, producing 2,850 core gene clusters found in four out of the five species. Integration of Tn-Seq fitness data enabled identification of 373 protein clusters that were conserved in all five species. A scoring rubric and filter was applied to these clusters, which incorporated Tn-Seq fitness defects, operon localization, and antibiotic susceptibility data, which reduced the number of bacteremia-fitness genes and identified seven common fitness mechanisms. Independent mutational validation of one prioritized fitness gene, tatC, showed reduced fitness in vivo and increased susceptibility to beta- lactams that were restored following tatC complementation in trans. By integrating known operon structures and antibiotic susceptibility with Tn-Seq fitness data, common genes within the core pan-genome emerged and revealed mechanisms that are essential for colonization of, or survival in, the mammalian bloodstream. Our prediction and validation of tatC as a common bacteremia fitness factor and contributor of antibiotic resistance supports the utility of this bioinformatic approach. This study represents a major step forward to prioritize potentially novel targets for therapy against these deadly widespread sepsis infections. Author SummaryBacteremia is a leading cause of sepsis, a life-threatening condition where an unregulated immune response to infection causes systemic organ failure. Nearly half of bacteremia cases are caused by members of the Gram-negative bacterial taxonomic order Enterobacterales. Given the public health impact of bacteremia and the reduction of existing antibiotic treatment options, novel strategies are needed to combat these infections. Pan-genome software was used to predict seven shared fitness pathways in these bacteria that may serve as novel targets for treatment of bacteremia. Briefly, a scoring rubric was applied to shared pan-genome clusters, which incorporated multiple data types, including Tn-Seq fitness defects, operon localization, and antibiotic susceptibility data to rank and prioritize fitness genes. To validate one of our predictions, mutations were constructed in tatC, which showed both reduced fitness in mice and increased susceptibility to beta-lactam antibiotics; complementation restored fitness and antibiotic susceptibility to wild type levels. This study takes a novel bioinformatics approach to build a core pan-genome across multiple distantly related bacteria to integrate computational and experimental data to predict important shared fitness genes and represents a major step forward toward identifying novel targets of therapy against these deadly widespread life-threatening infections.

bioinformatics↗

bioRxiv2023

Activation of a Vibrio cholerae CBASS anti-phage system by quorum sensing and folate depletion

A major challenge faced by bacteria is infection by bacteriophage (phage). Abortive infection is one strategy for combating phage in which an infected cell kills itself to limit phage replication, thus protecting neighboring kin. One class of abortive infection systems is the cyclic oligonucleotide based anti-phage signaling system (CBASS) which relies on two core enzymatic activities; an oligo-nucleotide cyclase that is activated following phage infection and a cyclic-oligo-nucleotide sensitive effector whose activity kills the infected cell. However, the mechanisms behind the deployment and activation of these lethal CBASS systems prior-to and following infection have largely remained a mystery. While exploring unique genomic features of the current pandemic Vibrio cholerae biotype El Tor for clues underlying its pandemic success we found its CBASS was spuriously activated by the folate biosynthesis inhibitor sulfamethoxazole, but only after the population had reached a high-cell density. This population density dependent activity revealed that transcription of both the oligo-nucleotide cyclase, dncV, and the CBASS phospholipase effector, capV, is enhanced at high-cell density by quorum sensing. Together, these results demonstrate that the V. cholerae CBASS is deployed when the environment is densely populated and activated in response to a perturbation in folate biosynthesis. SignificanceTo counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the 7th V. cholerae pandemic and more broadly how bacteria defend themselves against phage infection.

microbiology↗

bioRxiv2022

The ADP-heptose biosynthesis enzyme GmhB is a conserved Gram-negative bacteremia fitness factor

Klebsiella pneumoniae is a leading cause of Gram-negative bacteremia, which is a major source of morbidity and mortality worldwide. Gram-negative bacteremia requires three major steps: primary site infection, dissemination to the blood, and bloodstream survival. Since K. pneumoniae is a leading cause of healthcare-associated pneumonia, the lung is a common primary infection site leading to secondary bacteremia. K. pneumoniae factors essential for lung fitness have been characterized, but those required for subsequent bloodstream infection are unclear. To identify K. pneumoniae genes associated with dissemination and bloodstream survival, we performed insertion site sequencing (InSeq) using a pool of >25,000 transposon mutants in a murine model of bacteremic pneumonia. This analysis revealed the gene gmhB as important for either dissemination from the lung or bloodstream survival. In Escherichia coli, GmhB is a partially redundant enzyme in the synthesis of ADP-heptose for the lipopolysaccharide (LPS) core. To characterize its function in K. pneumoniae, an isogenic knockout strain ({Delta}gmhB) and complemented mutant were generated. During pneumonia, GmhB did not contribute to lung fitness and did not alter normal immune responses. However, GmhB enhanced bloodstream survival in a manner independent of serum susceptibility, specifically conveying resistance to spleen-mediated killing. In a tail-vein injection of murine bacteremia, GmhB was also required by K. pneumoniae, E. coli and Citrobacter freundii for optimal bloodstream survival. Together, this study identifies GmhB as a conserved Gram-negative bacteremia fitness factor that acts through LPS-mediated mechanisms to enhance bloodstream survival. IMPORTANCEKlebsiella pneumoniae frequently causes healthcare-associated infections including pneumonia and bacteremia. This is particularly concerning due to emerging antimicrobial resistance and the propensity for bacteremia to initiate sepsis, which has high mortality and is the most expensive hospital-treated condition. Defining mechanisms of bloodstream survival is critical to understanding the pathology of bacteremia and identifying novel targets for future therapies. In this study, we identified the K. pneumoniae enzyme GmhB as a bloodstream-specific fitness factor that enables the bacteria to survive in the spleen but is dispensable in the lung. Furthermore, GmhB is also needed by the related bacterial pathogens Escherichia coli and Citrobacter freundii to cause bacteremia. Conserved bacteremia fitness factors such a GmhB could be the basis for future therapeutics that would alleviate significant disease caused by from multiple diverse pathogens.

microbiology↗

bioRxiv2021

A Broadly Conserved Deoxycytidine Deaminase Protects Bacteria from Phage Infection

The El Tor biotype of Vibrio cholerae is responsible for perpetuating the longest cholera pandemic in recorded history (1961-current). The genomic islands VSP-1 and -2 are two understudied genetic features that distinguish El Tor from previous pandemics. To understand their utility, we calculated the co-occurrence of VSP genes across bacterial genomes. This analysis predicted the previously uncharacterized vc0175, herein renamed deoxycytidylate deaminase Vibrio (dcdV), is in a gene network with dncV, a cyclic GMP-AMP synthase involved in phage defense. DcdV consists of two domains, a P-loop kinase and a deoxycytidylate deaminase, that are required for the deamination of dCTP and dCMP, inhibiting phage predation by corrupting cellular nucleotide concentrations. Additionally, DcdV is post-translationally inhibited by a unique noncoding RNA encoded 5 of the dcdV locus. DcdV homologs are conserved in bacteria and eukaryotes and our results identify V. cholerae DcdV as the founding member of a previously undescribed bacterial phage defense system.

microbiology↗

bioRxiv2019

Surface Sensing Stimulates Cellular Differentiation in Caulobacter crescentus

Cellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium C. crescentus employs a specialized dimorphic life cycle consisting of two differentiated cell types. How environmental cues, including mechanical inputs such as contact with a surface, regulate this cell cycle remain unclear. Here, we find that surface sensing by the physical perturbation of retracting extracellular pilus filaments accelerates cell cycle progression and cellular differentiation. We show that physical obstruction of dynamic pilus activity by chemical perturbation or by a mutation in the outer membrane pilus pore protein, CpaC, stimulates early initiation of chromosome replication. In addition, we find that surface contact stimulates cell cycle progression by demonstrating that surface-stimulated cells initiate early chromosome replication to the same extent as planktonic cells with obstructed pilus activity. Finally, we show that obstruction of pilus retraction stimulates the synthesis of the cell cycle regulator, cyclic diguanylate monophosphate (c-di-GMP) through changes in the activity and localization of two key regulatory histidine kinases that control cell fate and differentiation. Together, these results demonstrate that surface contact and mechanosensing by alterations in pilus activity stimulate C. crescentus to bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a surface-associated lifestyle. SignificanceCells from all domains of life sense and respond to mechanical cues [1-3]. In eukaryotes, mechanical signals such as adhesion and surface stiffness are important for regulating fundamental processes including cell differentiation during embryonic development [4]. While mechanobiology is abundantly studied in eukaryotes, the role of mechanical influences on prokaryotic biology remains under-investigated. Here, we demonstrate that mechanosensing mediated through obstruction of the dynamic extension and retraction of tight adherence (tad) pili stimulates cell differentiation and cell cycle progression in the dimorphic -proteobacterium Caulobacter crescentus. Our results demonstrate an important intersection between mechanical stimuli and the regulation of a fundamental aspect of cell biology.

microbiology↗