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

Abstract

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 (ΔgmhB) 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.

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

“…Mutations in arnD render K. pneumoniae significantly less hypermucoviscous and reduce capsular polysaccharide production (S3 Fig, (30)). In active human serum, the arnD mutant was defective for growth, which was attributable to complement-mediated killing as heat inactivation of human serum restored normal arnD replication (Fig 3B-C, S6 Fig) . A similar pattern was observed for the control strain rfaH, which lacks capsular polysaccharide and is more susceptible to killing by active human serum (11,12). Interestingly, murine serum replication was enhanced in the absence of ArnD (Fig 3A The arnD mutant had a subtle yet significant fitness defect in liver homogenate and a dramatic fitness defect in splenic homogenate (Fig 4A).…”

“…To model the third phase of bacteremia, survival in the blood and filtering organs, an intravascular model was used with inoculation of mice via tail vein injection (Fig 1A). This model bypasses the first two phases of bacteremia, initial site infection and dissemination, and allows for direct examination of bloodstream fitness (12,29). To define potential experimental bottlenecks to the spleen in this model, a fitness-neutral mutant was competed against wild-type KPPR1 at ratios of 1:1, 1:5,000, and 1:10,000.…”

“…Media were supplemented with 40µg/mL kanamycin to select for transposon mutants and isogenic knockout strains, or with 50µg/mL chloramphenicol to select for strains containing the plasmid pACYC184 and its derivatives. All bacterial strains in this study are detailed in S3 Table, and primers are detailed in S4 Table . Complementation plasmids were generated as previously described (12). The complementation vector, pACYC184 was linearized by BamHI and HindIII digestion (New England Biolabs, Ipswitch, MA).…”

“…Accordingly, K. pneumoniae lung fitness mechanisms have been extensively described and include a wide variety of factors including capsular polysaccharide, branched chain amino acid synthesis, and production of citrate synthetase (10,11). K. pneumoniae mechanisms enhancing dissemination have been less thoroughly described but are likely partially dependent on ADP-heptose biosynthesis and siderophores (12,13). The host hypoxia-inducible factor 1α (HIF-1α) in lung epithelial cells also promotes dissemination, although interactions at this step are not well understood (14).…”

Regulation of Klebsiella pneumoniae mucoidy by the bacterial tyrosine kinase Wzc

“…The GmhA binding partner, GmhB, has recently been shown to be critical for K. pneumoniae dissemination to the spleen via the bloodstream. 39 No reports on the function of GmhA2 have been published and one can only speculate how this proteins regulates K. pneumoniae CPS production and mucoidy. It is certainly intriguing how a protein predicted to bind phosphosugars and regulate chromosomal replication alters Wzc-Y-P i , EPS anchoring to the cell surface, and mucoidy.…”