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

Abstract

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 supported liver fitness in vivo 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. ArnD, PurD, DsbA, SspA, and PdxA increased fitness across bacteremia phases and each demonstrated unique fitness dynamics within compartments in this model. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA, but not PdxA, 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.

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

“…h arn operon not found in E. hormaechei i pdxA competitive index for K. pneumoniae has been reported in our published work (8). In the spleen (Table 3), four mutants (tatC, ruvA, gmhB, xerC) were attenuated in all five species.…”

“…h arn genes not present in E. hormaechei i pdxA competitive index for K. pneumoniae has been reported in our published work (8). (11,13).…”

“…ArcA was found as a significant fitness factor in K. pneumoniae, C. freundii, S. marcescens, and E. hormaechei, but not E. coli. Indeed, arcA was the most attenuated fitness factor gene mutant in our K. pneumoniae Tn-Seq bacteremia screen (8). Notably, the original Tn-Seq screens did not predict arcA as a fitness gene in E. coli and this was validated by lack of attenuation in a specifically constructed arcA mutant.…”

“…None of these mutant constructs were more susceptible to H2O2 across any species. Previous literature demonstrated that the gene sspA is required for K. pneumoniae oxidative stress resistance (8), although this gene is not traditionally annotated as involved in this stress response (8). Oxidative stress resistance may be conveyed by other fitness genes, and each species likely has unique mechanisms to combat this response.…”

“…To that end, we previously constructed random transposon libraries or ordered transposon libraries in representative clinical isolates of five Gram-negative Enterobacterales species: E. coli CFT073 (7), K. pneumoniae KPPR1 (8,9), S. marcescens UMH9 (10), C. freundii UMH14 (11), and E. hormaechei UM_CRE_14 (Ottosen, in preparation). We conducted global Tn-Seq screens in the murine model of bacteremia by tail vein inoculation and identified putative fitness genes predicted to be required by each species to survive in the bloodstream and colonize the spleen.…”