Date Published: August 26, 2019
Publisher: Public Library of Science
Author(s): Jay Vornhagen, Yuang Sun, Paul Breen, Valerie Forsyth, Lili Zhao, Harry L. T. Mobley, Michael A. Bachman, Matthew C. Wolfgang.
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.
Extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae and carbapenem-resistant Enterobacteriaceae (CRE) pose a serious public health threat due to their extensive antibiotic resistance. Many ESBL and CRE infections are healthcare-associated infections (HAIs), meaning they occur in long-term healthcare facilities and hospitals. Klebsiella pneumoniae (Kp) is an environmentally ubiquitous member of the Enterobacteriaceae family that can acquire antibiotic resistance genes , and thus is a leading cause of ESBL-producing Enterobacteriaceae infections  and HAIs . Mortality rates in patients infected with antibiotic resistant Kp often exceed 40% . Disturbingly, reports of hypervirulent clones of Kp acquiring mobile antibiotic resistance genes are becoming more frequent [5,6], posing a significant global threat to human health. As the efficacy of antibiotics diminishes and therapeutic options for patients infected with antibiotic resistant strains of Kp become increasingly limited, a better understanding of how Kp establish productive infections is necessary for the development of novel diagnostics and interventions to combat these dangerous bacteria.
To comprehensively identify novel Lcn2-dependent Kp factors during lung infection, we employed a previously described transposon library in the Kp strain KPPR1 [26,27]. To this end, Lcn2+/+ and Lcn2-/- mice  were retropharyngeally inoculated with a pool of ~25,000 transposon mutants (S1 Fig). Twenty-four hours after inoculation total lung CFU were collected for DNA extraction and InSeq analysis, as previously described . After filtering, each sample had greater than 50 million reads corresponding to greater than 20,000 unique transposon insertions inside of open reading frames (S1 Data). To identify Lcn2-dependent Kp factors, the ratio of transposon insertion reads within each gene between the Lcn2+/+ and Lcn2-/- lung output pools (mean reads per gene were 169 and 156, respectively) was calculated to generate a fitness index. Over 1,600 genes were significantly enriched in either mouse background indicating a potential interaction with Lcn2, including entB, which is required to synthesize both enterobactin and the Lcn2-evading siderophore salmochelin (S1 Data). To limit our analysis, only genes with a significant fitness index greater or less than 3 standard deviations from the mean were considered Lcn2-dependent, leading to a final list of 43 candidate genes (Table 1, S1 Fig, S1 Data).
The ability of a bacterial cell to utilize available nutrients upon encountering a new environment, referred to as metabolic flexibility, is critical for its survival and success. Bacteria must control highly interconnected metabolic pathways that are quickly activated based on substrate availability in their local environment. Central carbon metabolism connects many pathways in the cell by providing carbon skeletons for biosynthesis of macromolecular building blocks and conversely represents convergence points for the catabolism of macromolecules. Central carbon metabolism is comprised of glycolysis, gluconeogenesis, the Entner-Doudoroff pathway, the pentose phosphate pathway, and the citric acid cycle. The research presented here identifies the citric acid cycle component, citrate (Si)-synthase (GltA), as a critical mediator of metabolic flexibility in Kp, and this metabolic flexibility drastically influences fitness during infection in a site-specific manner. Using multiple murine models of infection in Lcn2+/+ and Lcn2-/- backgrounds, we show that GltA is a fitness factor during lung infection by direct and hematogenous routes but is not necessary for bacteremia. Additionally, GltA is necessary for gut colonization, which frequently precedes infection [12,13]. The necessity of GltA is likely determined by the nutrient composition of each respective body site, specifically access to amino acids that the bacteria cannot otherwise synthesize de novo. This is supported by the observation of differential fitness of KPPR1ΔgltA in the Lcn2+/+ and Lcn2-/- lung, which have different endogenous levels of amino acids. Together, these data provide new insight into how Kp metabolic flexibility determines fitness during infection. While the contributions of specific metabolic processes, such as iron acquisition [24,25,35,36,54–57], nitrogen utilization through urease activity , allantoin metabolism , and psicose metabolism  in Kp pathogenesis have been explored, this study reveals a role for central metabolism and metabolic flexibility during Kp infection.