Biology subjects

Forsyth, V.

Publications and source records attributed to Forsyth, V..

3 recordsLinked to original sources

bioRxiv2023

Klebsiella pneumoniae causes bacteremia using factors that mediate tissue-specific fitness and resistance to oxidative stress

Gram-negative bacteremia is a major cause of global morbidity involving three phases of pathogenesis: initial site infection, dissemination, and survival in the blood and filtering organs. Klebsiella pneumoniae is a leading cause of bacteremia and pneumonia is often the initial infection. In the lung, K. pneumoniae relies on many factors like capsular polysaccharide and branched chain amino acid biosynthesis for virulence and fitness. However, mechanisms directly enabling bloodstream fitness are unclear. Here, we performed transposon insertion sequencing (TnSeq) in a tail-vein injection model of bacteremia and identified 58 K. pneumoniae bloodstream fitness genes. These factors are diverse and represent a variety of cellular processes. In vivo validation revealed tissue-specific mechanisms by which distinct factors support bacteremia. ArnD, involved in Lipid A modification, was required across blood filtering organs and supported resistance to soluble splenic factors. The purine biosynthesis enzyme PurD largely enhanced liver fitness and was required for replication in serum. PdxA, a member of the endogenous vitamin B6 biosynthesis pathway, optimized replication in serum and lung fitness. The stringent response regulator SspA was required for splenic fitness yet was dispensable in the liver. In a bacteremic pneumonia model that incorporates initial site infection and dissemination, splenic fitness defects were enhanced, and DsbA, SspA, and PdxA increased fitness across bacteremia phases. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA specifically resists oxidative stress produced by NADPH oxidase Nox2 in the lung, spleen, and liver, as it was a fitness factor in wild-type but not Nox2-deficient (Cybb-/-) mice. These results identify site-specific fitness factors that act during the progression of Gram-negative bacteremia. Defining K. pneumoniae fitness strategies across bacteremia phases could illuminate therapeutic targets that prevent infection and sepsis. Author SummaryGram-negative bacteremia is a deadly family of infections that initiate sepsis, a leading cause of global morbidity and mortality. Only a small number of Gram-negative species contribute to the majority of clinical bacteremia. Klebsiella pneumoniae is the second leading cause of Gram-negative bacteremia, and the third leading cause of overall bloodstream infection. K. pneumoniae is highly linked to hospital-associated infection with increasing antimicrobial resistance, endangering the most vulnerable patients. It is critical to understand the pathogenesis of K. pneumoniae bacteremia to better develop targets for future therapies that can prevent these deadly infections. Here, we define over 50 K. pneumoniae genes that support bloodstream fitness. These factors are diverse, support tissue-specific fitness, and increase bacterial resistance to oxidative stress. Our study is the first to systematically define K. pneumoniae factors enhancing bacteremia in a mammalian system. These results illuminate host-pathogen interactions during K. pneumoniae bacteremia that may be extended to additional Gram-negative species.

microbiology↗

bioRxiv2020

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness

Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been stymied by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying levels of CPS production and hmv, were all significantly out-competed by wildtype in a murine model of disseminating pneumonia. This suggests that an optimal balance between cellular energetics, CPS biosynthesis and hmv are required for maximal fitness. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that these processes are integrated into the metabolic status of the cell. Therefore, hvKp may require certain nutrients to fully elaborate its virulence-associated properties to specifically cause deep tissue infections. Author summaryKlebsiella pneumoniae is a common multi-drug resistant hospital-associated pathogen, however some isolates are capable of causing community-acquired infections in otherwise healthy individuals. The strains causing community-acquired infections have some distinguishing characteristics, which include overproduction of capsule and hypermucoviscosity. Hypermucoviscous strains are very tacky and sediment poorly when centrifuged. Historically, hypermucoviscosity has been attributed to overproduction of capsular polysaccharide, but recent data suggest that other factors contribute to this bacterial phenotype. Moreover, it seems that capsule and hypermucoviscosity may have distinct roles in pathogenesis. In this study, we sought to systematically investigate the genes that contribute to capsule and hypermucoviscosity. We found that in most cases, genes coordinately impact both capsule biosynthesis and hypermucoviscosity. Some metabolic genes linked to the TCA cycle, however, only affect one of these properties. Here, we identify that capsule biosynthesis and hypermucoviscosity are tightly tied to central metabolism and that an optimal balance between metabolism, capsule, and hypermucoviscosity are important for in vivo fitness of K. pneumoniae. These results identify genes that can be further probed to dissect how capsule and hypermucoviscosity are coordinated in response to niche-specific nutrients. Such studies will expand our understanding of the factors that drive the pathobiology of hypervirulent K. pneumoniae.

microbiology↗

bioRxiv2019

The Klebsiella pneumoniae citrate synthase gene, gltA, influences site specific fitness during infection

Klebsiella pneumoniae (Kp), one of the most common causes of healthcare-associated infections, increases patient morbidity, mortality and hospitalization costs. Kp must acquire nutrients from the host for successful infection. However, the host is able to prevent bacterial nutrient acquisition through multiple systems, including the innate immune protein lipocalin 2 (Lcn2) that prevents Kp iron acquisition by sequestering the siderophore enterobactin. To identify novel Kp factors that mediate evasion of nutritional immunity during lung infection, we undertook an InSeq study using a pool of >20,000 transposon mutants administered to Lcn2+/+ and Lcn2-/- mice. Comparing mutant frequencies between mouse genotypes, this genome-wide screen identified the Kp citrate synthase GltA as potentially interacting with Lcn2, and this novel finding was independently validated. Interestingly, in vitro studies suggest that this interaction is not direct. Given that GltA is involved in oxidative metabolism, we screened the ability of this mutant to use a variety of carbon and nitrogen sources. The results indicated that the gltA mutant has a distinct amino acid auxotrophy and is unable to use a variety of carbon sources. Specifically, we show that gltA is necessary for growth in bronchioloalveolar lavage fluid, which is amino acid-limited, but dispensable in serum, which is amino acid rich. Deletion of Lcn2 from the host leads to increased amino acid levels in bronchioloalveolar lavage fluid, and thus abrogates the loss of gltA during pneumonia in the Lcn2-/- background. GltA was also required for gut colonization, but dispensable in the bloodstream in a bacteremia model, demonstrating that deletion of gltA leads to an organ-specific fitness defect. Together, this study is the first to mechanistically describe a role for gltA in Kp infection and provide unique insight into how metabolic flexibility impacts bacterial fitness during infection.\n\nAuthor SummaryThe bacteria Klebsiella pneumoniae (Kp) is an important cause of infection in healthcare settings. These infections can be difficult to treat, as they frequently occur in chronically ill patients and the bacteria has the ability to acquire multiple antibiotic resistance markers. Kp is a common colonizer of the intestinal tract in hospitalized patients, and can progress to infections of the bloodstream, respiratory and urinary tract. However, the bacterial factors that allow Kp to replicate in these different body sites is unclear. In this study, we found that the Kp citrate synthase, GltA, enables bacterial replication in the lung and intestine by enhancing the ability of Kp to use diverse nutrients, in a mechanism known as metabolic flexibility. Kp lacking GltA require specific amino acids that are abundant in blood, but not other body sites. The work in this study provides novel insight into why Kp is a successful hospital pathogen that can colonize and infect multiple body sites.

microbiology↗