The polysaccharide capsule is an essential virulence factor for Klebsiella pneumoniae in both community-acquired hypervirulent strains as well as health care-associated classical strains that are posing significant challenges due to multidrug resistance. Capsule production is known to be transcriptionally regulated by a number of proteins, but very little is known about how these proteins collectively control capsule production. RmpA and RcsB are two known regulators of capsule gene expression, and RmpA is required for the hypermucoviscous (HMV) phenotype in hypervirulent K. pneumoniae strains. In this report, we confirmed that these regulators performed their anticipated functions in the ATCC 43816 derivative, KPPR1S: rcsB and rmpA mutants are HMV negative and have reduced capsule gene expression. We also identified a novel transcriptional regulator, RmpC, encoded by a gene near rmpA. The ΔrmpC strain has reduced capsule gene expression but retains the HMV phenotype. We further showed that a regulatory cascade exists in which KvrA and KvrB, the recently characterized MarR-like regulators, and RcsB contribute to capsule regulation through regulation of the rmpA promoter and through additional mechanisms. In a murine pneumonia model, the regulator mutants have a range of colonization defects, suggesting that they regulate virulence factors in addition to capsule. Further testing of the rmpC and rmpA mutants revealed that they have distinct and overlapping functions and provide evidence that HMV is not dependent on overproduction of capsule. This distinction will facilitate a better understanding of HMV and how it contributes to enhanced virulence of hypervirulent strains. IMPORTANCE Klebsiella pneumoniae continues to be a substantial public health threat due to its ability to cause health care-associated and community-acquired infections combined with its ability to acquire antibiotic resistance. Novel therapeutics are needed to combat this pathogen, and a greater understanding of its virulence factors is required for the development of new drugs. A key virulence factor for K. pneumoniae is the capsule, and community-acquired hypervirulent strains produce a capsule that causes hypermucoidy. We report here a novel capsule regulator, RmpC, and provide evidence that capsule production and the hypermucoviscosity phenotype are distinct processes. Infection studies showing that this and other capsule regulator mutants have a range of phenotypes indicate that additional virulence factors are in their regulons. These results shed new light on the mechanisms controlling capsule production and introduce targets that may prove useful for the development of novel therapeutics for the treatment of this increasingly problematic pathogen.

Klebsiella pneumoniae is widely recognized as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of hospital-acquired infections (urinary tract infections [UTI], pneumonia, sepsis) and community-acquired invasive infections. The genetic heterogeneity of K. pneumoniae isolates complicates our ability to understand the virulence of K. pneumoniae. Characterization of virulence factors conserved between strains as well as strain-specific factors will improve our understanding of this important pathogen. The MarR family of regulatory proteins is widely distributed in bacteria and regulates cellular processes such as antibiotic resistance and the expression of virulence factors. Klebsiella encodes numerous MarR-like proteins, and they likely contribute to the ability of K. pneumoniae to respond to and survive under a wide variety of environmental conditions, including those present in the human body. We tested loss-of-function mutations in all the marR homologues in a murine pneumonia model and found that two (kvrA and kvrB) significantly impacted the virulence of K1 and K2 capsule type hypervirulent (hv) strains and that kvrA affected the virulence of a sequence type 258 (ST258) classical strain. In the hv strains, kvrA and kvrB mutants displayed phenotypes associated with reduced capsule production, mucoviscosity, and transcription from galF and manC promoters that drive expression of capsule synthesis genes. In contrast, kvrA and kvrB mutants in the ST258 strain had no effect on capsule gene expression or capsule-related phenotypes. Thus, KvrA and KvrB affect virulence in classical and hv strains but the effect on virulence may not be exclusively due to effects on capsule production.

Pregnancy alters the disposition and exposure to multiple drugs indicated for pregnancy-related complications. Previous in vitro studies have shown that pregnancy-related hormones (PRHs) alter the expression and function of certain cytochrome P450s (CYPs) in human hepatocytes. However, the impact of PRHs on hepatic concentrations of non-CYP drug-metabolizing enzymes (DMEs) and transport proteins remain largely unknown. In this study, sandwich-cultured human hepatocytes (SCHH) from five female donors were exposed to vehicle or PRHs (estrone, estradiol, estriol, progesterone, cortisol, and placental growth hormone), administered individually or in combination, across a range of physiologically relevant PRH concentrations for 72 h. Absolute concentrations of 33 hepatic non-CYP DMEs and transport proteins were quantified in SCHH membrane fractions using a quantitative targeted absolute proteomics (QTAP) isotope dilution nanoLC-MS/MS method. The data revealed that PRHs altered the absolute protein concentration of various DMEs and transporters in a concentration-, isoform-, and hepatocyte donor-dependent manner. Overall, eight of 33 (24%) proteins exhibited a significant PRH-evoked net change in absolute protein concentration relative to vehicle control (ANOVA p < 0.05) across hepatocyte donors: 1/11 UGTs (9%; UGT1A4), 4/6 other DMEs (67%; CES1, CES2, FMO5, POR), and 3/16 transport proteins (19%; OAT2, OCT3, P-GP). An additional 8 (24%) proteins (UGT1A1, UGT2B4, UGT2B10, FMO3, OCT1, MRP2, MRP3, ENT1) exhibited significant PRH alterations in absolute protein concentration within at least two individual hepatocyte donors. In contrast, 17 (52%) proteins exhibited no discernable impact by PRHs either within or across hepatocyte donors. Collectively, these results provide the first comprehensive quantitative proteomic evaluation of PRH effects on non-CYP DMEs and transport proteins in SCHH and offer mechanistic insight into the altered disposition of drug substrates cleared by these pathways during pregnancy.

Infections caused by Klebsiella pneumoniae continue to be a global public health threat; the treatment of these infections is complicated by the high frequency of multidrug resistance. K. pneumoniae produces a polysaccharide capsule required for virulence. Hypervirulent isolates also have a hypermucoviscous (HMV) phenotype that increases virulence, and we recently demonstrated that a horizontally acquired gene, rmpD , is required for HMV and hypervirulence but that the identity of the polymeric product(s) in HMV isolates is uncertain.

Pregnancy-related hormones (PRH) are recognized as important regulators of hepatic cytochrome P450 enzyme expression and function. However, the impact of PRH on the hepatic expression and function of uridine diphosphate glucuronosyltransferases (UGTs) remains unclear. Using primary human hepatocytes, we evaluated the effect of PRH exposure on mRNA levels and protein concentrations of UGT1A1, UGT2B7, and other key UGT enzymes, and on the metabolism of labetalol (a UGT1A1 and UGT2B7 substrate commonly prescribed to treat hypertensive disorders of pregnancy). Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to the PRH estradiol, estriol, estetrol, progesterone, and cortisol individually or in combination. We quantified protein concentrations of UGT1A1, UGT2B7, and four additional UGT1A isoforms in SCHH membrane fractions and evaluated the metabolism of labetalol to its glucuronide metabolites in SCHH. PRH exposure increased mRNA levels and protein concentrations of UGT1A1 and UGT1A4 in SCHH. PRH exposure also significantly increased labetalol metabolism to its UGT1A1-derived glucuronide metabolite in a concentration-dependent manner, which positively correlated with PRH-induced changes in UGT1A1 protein concentrations. In contrast, PRH did not alter UGT2B7 mRNA levels or protein concentrations in SCHH, and formation of the UGT2B7-derived labetalol glucuronide metabolite was decreased following PRH exposure. Our findings demonstrate that PRH alter expression and function of UGT proteins in an isoform-specific manner and increase UGT1A1-mediated labetalol metabolism in human hepatocytes by inducing UGT1A1 protein concentrations. These results provide mechanistic insight into the increases in labetalol clearance observed in pregnant individuals.