Genome-Wide Identification of Klebsiella pneumoniae Fitness Genes during Lung Infection

Abstract

Klebsiella pneumoniae is an urgent public health threat because of resistance to carbapenems, antibiotics of last resort against Gram-negative bacterial infections. Despite the fact that K. pneumoniae is a leading cause of pneumonia in hospitalized patients, the bacterial factors required to cause disease are poorly understood. Insertion site sequencing combines transposon mutagenesis with high-throughput sequencing to simultaneously screen thousands of insertion mutants for fitness defects during infection. Using the recently sequenced K. pneumoniae strain KPPR1 in a well-established mouse model of pneumonia, insertion site sequencing was performed on a pool of >25,000 transposon mutants. The relative fitness requirement of each gene was ranked based on the ratio of lung to inoculum read counts and concordance between insertions in the same gene. This analysis revealed over 300 mutants with at least a 2-fold fitness defect and 69 with defects ranging from 10- to >2,000-fold. Construction of 6 isogenic mutants for use in competitive infections with the wild type confirmed their requirement for lung fitness. Critical fitness genes included those for the synthesis of branched-chain and aromatic amino acids that are essential in mice and humans, the transcriptional elongation factor RfaH, and the copper efflux pump CopA. The majority of fitness genes were conserved among reference strains representative of diverse pathotypes. These results indicate that regulation of outer membrane components and synthesis of amino acids that are essential to its host are critical for K. pneumoniae fitness in the lung.

Copper Is a Host Effector Mobilized to Urine during Urinary Tract Infection To Impair Bacterial Colonization

“…While our work demonstrates urinary Cu mobilization via ceruloplasmin during UTI with Gram-negative pathogens, whether the Cu mobilization response is conserved during UTI with Gram-positive pathogens remains to be examined. Mutants lacking Cu detoxification systems in diverse pathogens, including Cryptococcus neoformans (a fungus), K. pneumoniae, Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, Streptococcus pneumoniae, and UPEC, are attenuated in animal models of infection (11,(26)(27)(28)(29)(30)(31). Our results presented here elucidate the protective role of Cu in the pathogenesis of UTI.…”

Klebsiella pneumoniae: Going on the Offense with a Strong Defense

“…Furthermore, it is important to remember that the majority of people presenting with K. pneumoniae pneumonia and bacteremia are in hospitals or long-term-care facilities and are immunosuppressed in some manner. Many of the recent searches for virulence factors have focused on identifying virulence factors in "normal" mice; however, a subset of these factors may not be required in immunosuppressed environments (363). In fact, different genes may play a role in immunosuppressed patient subsets that have yet to be identified and characterized.…”

“…In this study, a preliminary characterization of 6 of these genes found that they appear to be important for virulence by playing either a protective role against the immune response or a role in the acquisition of resources. Specifically, those authors found that rfaH promoted serum resistance and capsule production, aroE promoted serum resistance, ilvC and ilvD were important for branched-chain-amino-acid synthesis, and copA promoted resistance to copper toxicity (363).…”

“…Specifically, two screens were performed to identify K. pneumoniae genes critical for survival in the lung, two were performed for genes required for intestinal colonization (with one of these screens also looking at UTIs), and one screen was performed in a liver abscess model (167,(363)(364)(365)(366). The majority of these screens were performed with classical K. pneumoniae strains, although one screen by Tu et al focused on an HV K. pneumoniae strain (366).…”

Using bacterial genomes and essential genes for the development of new antibiotics

“…The authors then used transposon-directed insertion site sequencing to identify approximately 400 genes that were essential for growth of C. difficile in vitro and 798 genes that participate in C. difficile sporulation. In a separate study, more than 25,000 random insertion mutants were generated in the Klebsiella pneumniae reference strain KPPR1 and genes that participate in establish lung infection in a mouse model were identified by comparing the relative abundance of mutants in the initial inoculum and in the lung after infection [55]. This study identified more than 300 genes that resulted in at least a two-fold reduction in abundance in lung tissue when compared to the inoculum, and 69 genes with at least a 10-fold decrease in abundance.…”