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

Abstract

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. This includes the innate immune protein lipocalin 2 (Lcn2), which prevents Kp iron acquisition. To identify novel Lcn2-dependent 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 transposon mutant frequencies between mouse genotypes, we 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 rendering it reliant upon glutamate family amino acids for growth. Deletion of Lcn2 from the host leads to increased amino acid levels in bronchioloalveolar lavage fluid, corresponding to increased fitness of the gltA mutant in vivo and ex vivo . Accordingly, addition of glutamate family amino acids to Lcn2 +/+ bronchioloalveolar lavage fluid rescued growth of the gltA mutant. Using a variety of mouse models of infection, we show that GltA is an organ-specific fitness factor required for complete fitness in the spleen, liver, and gut, but dispensable in the bloodstream. Similar to bronchioloalveolar lavage fluid, addition of glutamate family amino acids to Lcn2 +/+ organ lysates was sufficient to rescue the loss of gltA . Together, this study describes a critical role for GltA in Kp infection and provides unique insight into how metabolic flexibility impacts bacterial fitness during infection.

Replication Dynamics for Six Gram-Negative Bacterial Species during Bloodstream Infection

“…The systemic nature of BSI provides numerous local environments for bacteria to establish a replicative niche. Previous work has established that large numbers of bacterial cells reside in the spleens, livers, and kidneys of mice infected via the tail vein route ( 18 , 19 , 21 , 23 , 34 , 35 ). To determine the relevant infection niches for each of our six species of interest, bacteria were introduced into the bloodstream of mice and the numbers of viable bacteria in the spleen, liver, and kidneys were tracked over time.…”

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

“…Primers were designed using the online NEBuilder Assembly Tool with the following settings: >20 nucleotide overlap, Phusion DNA Polymerase (HF Buffer), 500 nM primer concentration ( S1 Table ). The ORF and 500 bp of the predicted promoter region were exchanged with 600 bp of the tet cassette or 200 bp of the cat cassette in pACYC184 [ 67 ]. Gel purified PCR products were assembled according to the manufacturer’s instructions and the enzymatic reaction was incubated at 50°C for 1 h. The NEBuilder reaction was dialyzed overnight against 10% sterile glycerol using a VSWF 0.025 μm filter disk.…”

An acquired acyltransferase promotes Klebsiella pneumoniae ST258 respiratory infection

“…Within CRKP, the sequence type 258 (ST258) has emerged as a persistent and overrepresented cause of hospital-associated infections in the United States and worldwide (Marsh et al, 2019;Tzouvelekis et al, 2013;van Duin et al, 2020). Transposon screens have been used to identify loci that promote K. pneumoniae pathogenicity (Bachman et al, 2015;Paczosa et al, 2020;Vornhagen et al, 2019). However, such studies typically use a laboratory reference isolate (American Type Culture Collection [ATCC] 43816 or KPPR1), which is genetically distinct from the highly anti-biotic resistant ST258 clonal complex (Gomez-Simmonds and Uhlemann, 2017;Vornhagen et al, 2019).…”

“…Transposon screens have been used to identify loci that promote K. pneumoniae pathogenicity (Bachman et al, 2015;Paczosa et al, 2020;Vornhagen et al, 2019). However, such studies typically use a laboratory reference isolate (American Type Culture Collection [ATCC] 43816 or KPPR1), which is genetically distinct from the highly anti-biotic resistant ST258 clonal complex (Gomez-Simmonds and Uhlemann, 2017;Vornhagen et al, 2019). To understand the bacterial properties responsible for the success of K. pneumoniae ST258, we analyzed clinical isolate 35 (KP35) from our own hospital, a representative strain that expresses the most common cps (capsular protein) and wzi (capsular component) genotypes (Ahn et al, 2016;Gomez-Simmonds et al, 2015).…”

“…Comprehensive genotypic and phenotypic studies of ST258 isolates have identified interesting but inconsistent virulence determinants such as those involved in iron utilization, stress response signaling, biofilm formation, and type IV pili (Marsh et al, 2019;Pitout et al, 2015). In the absence of a unifying virulence phenotype, it has been postulated that the dynamic evolution of these mechanisms of persistence within the host microbiota is due to the availability of metabolites generated by recruited immune cells as well as by bacteria (Blin et al, 2017;Vornhagen et al, 2019). We hypothesized that the success of pathogens like ST258 K. pneumoniae could also be attributable to less apparent factors such as the metabolic properties of the bacteria.…”