Contributions of TolC Orthologs to Francisella tularensis Schu S4 Multidrug Resistance, Modulation of Host Cell Responses, and Virulence

Abstract

Francisella tularensis is a Gram-negative, facultative intracellular pathogen and the causative agent of tularemia. Previous studies with the attenuated live vaccine strain (LVS) identified a role for the outer membrane protein TolC in modulation of host cell responses during infection and virulence in the mouse model of tularemia. TolC is an integral part of efflux pumps that export small molecules and type I secretion systems that export a range of bacterial virulence factors. In this study, we analyzed TolC and its two orthologs, FtlC and SilC, present in the fully virulent F. tularensis Schu S4 strain for their contributions to multidrug efflux, suppression of innate immune responses, and virulence. We found that each TolC ortholog participated in multidrug efflux, with overlapping substrate specificities for TolC and FtlC and a distinct substrate profile for SilC. In contrast to their shared roles in drug efflux, only TolC functioned in the modulation of macrophage apoptotic and proinflammatory responses to Schu S4 infection, consistent with a role in virulence factor delivery to host cells. In agreement with previous results with the LVS, the Schu S4 ΔtolC mutant was highly attenuated for virulence in mice by both the intranasal and intradermal routes of infection. Unexpectedly, FtlC was also critical for Schu S4 virulence, but only by the intradermal route. Our data demonstrate a conserved and critical role for TolC in modulation of host immune responses and Francisella virulence and also highlight strain-and route-dependent differences in the pathogenesis of tularemia.

Genetic Determinants of Antibiotic Resistance in Francisella

“…However, this annotation as being important in cationic antimicrobial peptide resistance has not been experimentally confirmed for any of these. tolC mutants had the same polymyxin sensitivity as wild-type F. tularensis SchuS4 (Kopping et al, 2019). Experiments examining the importance of AcrA/B/TolC in F. tularensis LVS have also not demonstrated a role for these genes in polymyxin resistance (Gil et al, 2006;Kopping et al, 2019).…”

“…tolC mutants had the same polymyxin sensitivity as wild-type F. tularensis SchuS4 (Kopping et al, 2019). Experiments examining the importance of AcrA/B/TolC in F. tularensis LVS have also not demonstrated a role for these genes in polymyxin resistance (Gil et al, 2006;Kopping et al, 2019). LpxA, the LPS biosynthesis gene FTT_1569c [FTN1478], was annotated by KEGG as playing a role in cationic antimicrobial peptide resistance and interacting with AcrA/B/TolC.…”

“…It appears that EmrA1 is involved with resistance to streptomycin and neomycin but not gentamicin ( Ma et al, 2014 ), as well as SDS ( Enstrom et al, 2012 ) in Francisella . The relationship and interchangeability of the various TolC orthologs in Francisella are more thoroughly discussed in Kopping et al (2019) . The various pumps/membrane proteins/fusion proteins in Francisella are illustrated in Figure 1 .…”

“…Francisella species contain genes for very few efflux pumps ( Table 4 and Figure 1 ). Overall, there are three closely related pumps, classified as Type I secretion systems, including the TolC/AcrA/B efflux pump ( Kopping et al, 2019 ), the TolC homolog FtlC ( Gil et al, 2006 ), as well as SilC and the HylD/EmrA1 system ( Ma et al, 2014 ; Alqahtani et al, 2018 ). The TolC/AcrA/AcrB complex is an important and well-characterized multidrug-resistant efflux pump in Francisella ( Kopping et al, 2019 ).…”

“…Overall, there are three closely related pumps, classified as Type I secretion systems, including the TolC/AcrA/B efflux pump ( Kopping et al, 2019 ), the TolC homolog FtlC ( Gil et al, 2006 ), as well as SilC and the HylD/EmrA1 system ( Ma et al, 2014 ; Alqahtani et al, 2018 ). The TolC/AcrA/AcrB complex is an important and well-characterized multidrug-resistant efflux pump in Francisella ( Kopping et al, 2019 ). As described in the sections above, the TolC/AcrA/AcrB efflux pump is shown to play important roles in erythromycin, SDS ( Kopping et al, 2019 ), and aminoglycoside resistance ( Gil et al, 2006 ).…”