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

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.
Author summary The bacteria Klebsiella pneumoniae (Kp) is an important cause of infection in healthcare settings. These infections can be difficult to treat, as they frequently occur in chronically ill patients and the bacteria have the ability to acquire multiple antibiotic resistance markers. Kp is a common colonizer of the intestinal tract in hospitalized patients, and can progress to infections of the bloodstream, respiratory, and urinary tract. However, the bacterial factors that allow Kp to replicate in these different body sites are unclear. In this study, we found that the Kp citrate synthase, GltA, enables bacterial replication in the lung and intestine by enhancing the ability of Kp to use diverse nutrients in a mechanism known as metabolic flexibility. Kp lacking GltA require specific amino acids that are abundant in blood, but not other body sites. The work in this study provides novel insight into why Kp is a successful hospital pathogen that can colonize and infect multiple body sites.