Antibiotic-resistant strains of Klebsiella pneumoniae often exhibit porin loss. In this study, we investigated how porin loss impacted the composition of secreted outer membrane vesicles as well as their ability to trigger proinflammatory cytokine secretion by macrophages. We hypothesize that porin loss associated with antibiotic resistance will directly impact both the composition of outer membrane vesicles and their interactions with phagocytic cells. Using clonally related clinical isolates of extended-spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae with different patterns of porin expression, we demonstrated that altered expression of OmpK35 and OmpK36 results in broad alterations to the protein profile of secreted vesicles. Additionally, the level of OmpA incorporation was elevated in strains lacking a single porin. Porin loss significantly impacted macrophage inflammatory responses to purified vesicles. Outer membrane vesicles lacking both OmpK35 and OmpK36 elicited significantly lower levels of proinflammatory cytokine secretion than vesicles from strains expressing one or both porins. These data demonstrate that antibiotic resistance-associated porin loss has a broad and significant effect on both the composition of outer membrane vesicles and their interactions with phagocytic cells, which may impact bacterial survival and inflammatory reactions in the host.K lebsiella pneumoniae is a nosocomial pathogen responsible for as many as 20% of all cases of culture-positive blood cultures and bacterial sepsis (1). Alarmingly, up to 50% of these infections are resistant to most current antibiotics (2, 3). The majority of resistant Klebsiella isolates exhibit both expression of an extendedspectrum beta-lactamase (ESBL) and halted expression of at least one outer membrane porin (4, 5). Porin loss has been clearly shown to enhance levels of antibiotic resistance, and ESBL-positive K. pneumoniae isolates commonly exhibit loss of OmpK35 and OmpK36, which both function in nonspecific transport across the outer membrane (6-8).While changes to the porin profile of bacteria are well documented to be associated with enhanced antibiotic resistance (9, 10), the role that these porins play in pathogenesis has not been fully investigated. In vitro experiments using mutants of K. pneumoniae lacking OmpK36 have demonstrated that loss of OmpK36 results in increased phagocytic killing of the bacteria, and in vivo experiments have demonstrated decreased virulence and bacterial survival of strains lacking OmpK36 in a mouse model (11)(12)(13)(14). Data from these studies consistently indicate that loss of OmpK36 results in an increase in antibiotic resistance paired with a decrease in fitness and ability to survive host immune responses. It is unclear how resistant strains of Klebsiella are able to persist in host tissues when porin loss renders the bacteria more susceptible to phagocytic clearance.The capsule is the best understood of the virulence factors of Klebsiella, cloaking the bacterial outer surface from immu...

Bacterial pathogens require a variety of micronutrients for growth, including trace metals such as iron, manganese, and zinc (Zn). Despite their relative abundance in host environments, access to these metals is severely restricted during infection due to host-mediated defense mechanisms collectively known as nutritional immunity. Despite a growing appreciation of the importance of Zn in host-pathogen interactions, the mechanisms of Zn homeostasis and the significance of Zn to the pathophysiology of E. faecalis , a major pathogen of nosocomial and community-associated infections, have not been thoroughly investigated. Here, we show that E. faecalis encoded ABC-type transporter AdcACB and an orphan substrate-binding lipoprotein AdcAII that work cooperatively to maintain Zn homeostasis. Simultaneous inactivation of adcA and adcAII or the entire adcACB operon led to a significant reduction in intracellular Zn under Zn-restricted conditions and heightened sensitivity to Zn-chelating agents including human calprotectin, aberrant cell morphology, and impaired fitness in serum ex vivo . Additionally, inactivation of adcACB and adcAII significantly reduced bacterial tolerance toward cell envelope-targeting antibiotics. Finally, we showed that the AdcACB/AdcAII system contributes to E. faecalis virulence in a Galleria mellonella invertebrate infection model and in two catheter-associated mouse infection models that recapitulate many of the host conditions associated with enterococcal human infections. Collectively, this report reveals that high-affinity Zn import is important for the pathogenesis of E. faecalis establishing the surface-associated AdcA and AdcAII lipoproteins as potential therapeutic targets.

Bacterial pathogens require a variety of micronutrients for growth, including trace metals such as iron, manganese, and zinc (Zn). Despite their relative abundance in host environments, access to these metals is severely restricted during infection due to host-mediated defense mechanisms collectively known as nutritional immunity. Despite a growing appreciation of the importance of Zn in host-pathogen interactions, the mechanisms of Zn homeostasis and the significance of Zn to the pathophysiology of E. faecalis, a major pathogen of nosocomial and community-associated infections, have not been investigated. Here, we show that E. faecalis encoded an ABC-type transporter AdcACB and an orphan substrate-binding lipoprotein AdcAII that work cooperatively to maintain Zn homeostasis. Simultaneous inactivation of adcA and adcAII or the entire adcACB operon led to significant reduction in intracellular Zn under Zn-restricted conditions, heightened sensitivity to Zn-chelating agents including human calprotectin, aberrant cell morphology, and impaired fitness in serum ex vivo. Additionally, inactivation of adcACB and adcAII significantly reduced bacterial tolerance towards cell envelope-targeting antibiotics, which may be associated to altered fatty acid abundance and species. Lastly, we show that the AdcACB/AdcAII system contributes to E. faecalis virulence in an invertebrate (Galleria mellonella) infection model and in two catheter-associated mouse infection models that recapitulate many of the host conditions associated with enterococcal human infections. Collectively, this report reveals that high-affinity Zn import is essential for the pathogenesis of E. faecalis indicating that the surface-associated AdcA and AdcAII lipoproteins are potential therapeutic targets.

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