Porin Loss Impacts the Host Inflammatory Response to Outer Membrane Vesicles of Klebsiella pneumoniae

Abstract

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...

Outer membrane vesicles from β-lactam-resistant Escherichia coli enable the survival of β-lactam-susceptible E. coli in the presence of β-lactam antibiotics

“…The β-lactam antibiotic must pass through the traverse porin channel in the outer membrane to access the PBP of the inner plasma membrane. However, proteins produced by mutations in porin-encoding genes or porin loss show lower permeability to β-lactam antibiotics 42 , 43 . Efflux pumps can export a wide range of substrates from the periplasm to the surrounding environment 44 , thus overproduction of efflux pump can augment the resistance of β-lactam antibiotics 45 .…”

Insertion Sequence-Dependent <i>OmpK</i>36 Mutation Associated Ertapenem Resistance in Clinical <i>Klebsiella pneumoniae</i>

“…In our study, ESBL-producing isolates and AmpC enzyme-producing isolates exhibited different resistance patterns with higher resistance rate to ceftriaxone and cefepime vs. higher resistance rates to ceftazidime and cefoxitin, respectively ( Table 2). Regardless of whether the isolate was ESBL-or non-ESBL-producing, the carbapenem-resistant isolates carried bla DHA-1 and an insertion in both ompK35 and ompK36 genes, exhibiting the highest MIC to carbapenems ( and a significant decrease in proinflammatory cytokine levels [45]. Further, the loss of OmpK35 is associated with resistance to lipophilic (benzylpenicillin) and large (cefepime) compounds [46].…”

Fine-tuning carbapenem resistance by reducing porin permeability of bacteria activated in the selection process of conjugation

“…In K. pneumoniae , loss of porins OmpK 35 and OmpK 36, homologs of OmpC and OmpF, respectively, showed different protein composition of outer membrane vesicles. These vesicles were found to induce secretion of extremely low levels of proinflammatory cytokine 50 . Indeed, low cytokine secretion may be an evasion mechanism for bacteria to escape from the host immune response.…”