Biology subjects

Anderson, M. T.

Publications and source records attributed to Anderson, M. T..

5 recordsLinked to original sources

bioRxiv2024

Infection characteristics among Serratia marcescens capsule lineages.

Serratia marcescens is a healthcare-associated pathogen that can cause severe infections including bacteremia and pneumonia. The capsule polysaccharide of S. marcescens is a bacteremia fitness determinant and previous work defined capsule locus (KL) diversity within the species. Strains belonging to KL1 and KL2 capsule clades produce sialylated polysaccharides and represent the largest subpopulation of isolates from clinical origin. In this study, the contribution of these and other S. marcescens capsules to infection was determined in animal and cellular models. Using a murine model of primary bacteremia, clinical isolates of multiple KL types demonstrated capsule-dependent colonization of spleen, liver, and kidney following tail vein inoculation. Similar results were observed using a bacteremic pneumonia model, in that all tested strains of clinical origin demonstrated a requirement for capsule in both the primary lung infection site and for bloodstream dissemination to secondary organs. Finally, capsule from each KL clade was examined for the ability to resist internalization by bone marrow-derived macrophages. Only the sialylated KL1 and KL2 clade strains exhibited capsule-dependent inhibition of internalization, including KL2 capsule produced in a heterologous background. Together these findings indicate that lineage-specific resistance to macrophage phagocytosis may enhance survival and antibacterial defenses of clinically-adapted S. marcescens. IMPORTANCEBacteremia occurs when the host immune system fails to contain bacterial bloodstream replication following an initial inoculation event from either an internal or external source. Capsule polysaccharides play a protective role for Serratia marcescens during bacteremia but there is abundant genetic diversity at the capsule-encoding locus within the species. This study compares the infection characteristics of S. marcescens isolates belonging to five capsule types and defines the contributions to infection fitness for each. By characterizing the differences in capsule dependence and infection potential between S. marcescens strains, efforts to combat these life-threatening infections can be focused toward identifying strategies that target the most critical genetic lineages of this important opportunistic pathogen.

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↗

bioRxiv2024

Fitness Factor Genes Conserved within the Multi-species Core Genome of Gram-negative Enterobacterales Species Contribute to Bacteremia Pathogenesis

There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from an unregulated immune response to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Each mutant in each species was used to cochallenge C57BL/6 mice via tail vein injection along with the respective wild-type strain to determine competitive indices for each fitness gene or operon. Among the five species, we found three fitness factor genes, that when mutated, attenuated the mutant for all species in the spleen and liver (tatC, ruvA, gmhB). Nine additional fitness factor genes or operons were validated as outcompeted by wild-type in three or four bacterial species in the spleen (xerC, wzxE, arcA, prc, apaGH, atpG, lpdA, ubiH, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections. >Author SummaryFrequent cases of bacteremia plague our ICUs, bone marrow transplant units, and inpatient facilities. Nearly half of these infections are caused by Gram-negative bacteria. The Enterobacterales order including E. coli, K. pneumoniae, S. marcescens, C. freundii, and E. hormaechei are leading causes of bacteremia. An alarming proportion of these are due to antibiotic-resistant isolates, which are four times more likely to kill than antibiotic-susceptible isolates. Clearly, we need new therapeutic targets to treat cases of bacteremia and sepsis. Previously, it has been unclear what genes contribute to their ability to survive in this hostile host environment. We have previously undertaken unbiased genetic screens to identify 18 genes shared by all five bacterial genera that are required for survival in blood and blood-filtering organs. These include genes that encode proteins that maintain proton motive force, resist antimicrobial peptides and complement, mediate genome maintenance, transport key metabolites and proteins, avoid oxidative stress, acquire iron, and regulate key pathways. Mutants, constructed in these shared genes in the five species, were validated for a high proportion of genes as critical for infection in the mouse model of bacteremia.

microbiology↗

bioRxiv2023

Tor1 dysfunction promotes phenotypic diversity in the asexual fungus Candida albicans

Genetic variation is a primary contributor to phenotypic variation within a population. In asexual eukaryotes however, it is unclear how, or if, genetic variation is generated and maintained to promote phenotypic variation. C. albicans, an asexual fungus that causes opportunistic infections in susceptible hosts, has several phenotypic switching systems, including the colony morphology phenotypic switching (CMPS) system. CMPS, a penetrant change in colony morphology on solid medium in vitro, is associated with incipient or fulminant clinical disease. CMPS results in the alteration of additional virulence properties, including drug resistance, that are not tightly correlated with colony morphology. Importantly, it is unknown whether CMPS is a regulated or stochastic process. We found that specific mutants affecting the Target of Rapamycin (TOR) pathway showed an increase in CMPS frequency and CMPS in these mutant backgrounds was associated with changes in rapamycin sensitivity. We also identified growth conditions that promoted CMPS in clinical strains and found that CMPS in these backgrounds was also linked to the TOR pathway through changes in rapamycin sensitivity. These results demonstrate that CMPS promotes phenotypic variation through the TOR pathway, supporting a model that this is a regulated process. Since the TOR growth control pathway is conserved throughout the eukarya, the identification of TOR as a phenotypic diversity regulator likely has broad implications.

genetics↗

bioRxiv2023

Conserved metabolic regulator ArcA responds to oxygen availability, iron limitation, and cell envelope perturbations during bacteremia

Bacteremia, a systemic infection associated with severe clinical outcomes, is often caused by Gram-negative facultative anaerobes. ArcAB, a two-component regulatory system that represses aerobic respiration, is a key mediator of metabolic adaptation for such bacteria. Using targeted mutational analysis informed by global genetic screens, we identified the arcA gene as promoting fitness of Klebsiella pneumoniae, Citrobacter freundii, and Serratia marcescens but not Escherichia coli in a murine model of bacteremia. Engineered mutants lacking arcA exhibit a dysregulated response to changes in oxygen availability, iron limitation, and membrane perturbations, all of which bacterial cells experience during infection. The genetic response of the arcA mutants relative to wild-type strains to the cationic antimicrobial peptide polymyxin B demonstrates an expanded role for ArcA as an activator in response to membrane damage in addition to metabolic adaptation. ArcA function is furthermore linked to electron transport chain activity based on its response to uncoupling of proton motive force by carbonyl cyanide-m-chlorophenylhydrazone (CCCP). Differences in lactate and acetate levels as well as lactate dehydrogenase activity between arcA mutant and wild-type cells following CCCP treatment establish an ArcA-mediated shift to fermentation independent of oxygen availability. This study highlights the semi-conserved role of ArcA during bacteremia and consolidates infection phenotypes into a comprehensive model based on respiratory activity. AUTHOR SUMMARYInfections of the bloodstream are life-threatening and can result in sepsis, an overreaction of the host immune system that ultimately damages the body. Gram-negative bacteria are responsible for causing many cases of bloodstream infections, also referred to as bacteremia. The long-term goal of our work is to understand how these bacteria establish and maintain infection during bacteremia. We have previously identified the transcription factor ArcA, which promotes fermentation in bacteria, as a likely contributor to the growth and survival of bacteria in this environment. Here, we study ArcA in the Gram-negative species Citrobacter freundii, Klebsiella pneumoniae, and Serratia marcescens. Our findings aid in determining how these bacteria sense their environment, utilize nutrients, and generate energy while also countering attacks from the host immune system. This information is critical for developing better models of infection to inform future therapeutic development.

microbiology↗