Klebsiella pneumoniaecauses 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 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.

Conserved Metabolic Regulator ArcA Responds to Oxygen Availability, Iron Limitation, and Cell Envelope Perturbations during Bacteremia

“…S. marcescens arcA mutant was most outcompeted in the kidneys (33.7-fold), together indicating the magnitude of ArcA's contribution to fitness in this model is organ-and species-specific. These results validate and confirm our previous TnSeq findings that initially identified the fitness potential of ArcA among a vast pool of transposon mutants (10)(11)(12). No significant fitness defect was observed for the E. coli arcA mutant in either the spleen or the liver, a notable contrast to the other species.…”

“…Our group has previously utilized transposon mutant libraries and TnSeq to identify critical fitness genes for C. freundii, E. coli, K. pneumoniae, and S. marcescens during bacteremia (9)(10)(11)(12).…”

Patterns of Klebsiella pneumoniae bacteremic dissemination from the lung

“…TatC is part of the twin-arginine transporter required for moving multiple folded protein substrates across the cytoplasmic membrane (31). Unlike GmhB, which is dispensable in the lung, TatC is required for both primary and secondary site fitness during bacteremia in multiple species (32, 33). Based on bacterial burden, it is unclear if either is involved in dissemination.…”

“…To determine how host defenses determine the patterns of dissemination, we performed STAMPR analysis of wild-type K. pneumoniae in mice defective in NADPH oxidase Nox2 ( Cybb -/- ), and the monocyte chemokine receptor Ccr2 ( Ccr2 -/- ), which are required for the phagocyte oxidative burst and monocyte recruitment, respectively. Nox2 restricts K. pneumoniae replication in the lung but has more subtle influences in the spleen and liver (32). After lung infection, all Nox2 -/- mice exhibited direct dissemination, suggesting that phagocyte NADPH oxidase is required for the metastatic pattern of K. pneumoniae dissemination (Figure 3E-F).…”

“…Inflammatory reprogramming could change the bacterial ability to disseminate or influence fitness at secondary sites. For example, DsbA, involved in disulfide bond formation, is required for splenic fitness after tail vein injection but is dispensable when infection originates in the lung (32). Potentially during metastatic dissemination, bacteria that replicate in the lung are primed for increased splenic survival.…”