Assessing the Contribution of an HtrA Family Serine Protease During Borrelia turicatae Mammalian Infection

Abstract

Tick-borne relapsing fever (TBRF), characterized by recurring febrile episodes, is globally distributed and among the most common bacterial infections in some African countries. Despite the public health concern that this disease represents, little is known regarding the virulence determinants required by TBRF Borrelia during infection. Because the chromosomes of TBRF Borrelia show extensive colinearity with those of Lyme disease (LD) Borrelia , the exceptions represent unique genes encoding proteins that are potentially essential to the disparate enzootic cycles of these two groups of spirochetes. One such exception is a gene encoding an HtrA family protease, BtpA, that is present in TBRF Borrelia , but not in LD spirochetes. Previous work suggested that btpA orthologs may be important for resistance to stresses faced during mammalian infection. Herein, proteomic analyses of the TBRF spirochete, Borrelia turicatae , demonstrated that BtpA, as well as proteins encoded by adjacent genes in the B. turicatae genome, were produced in response to culture at mammalian body temperature, suggesting a role in mammalian infection. Further, transcriptional analyses revealed that btpA was expressed with the genes immediately upstream and downstream as part of an operon. To directly assess if btpA is involved in resistance to environmental stresses, btpA deletion mutants were generated. btpA mutants demonstrated no growth defect in response to heat shock, but were more sensitive to oxidative stress produced by t- butyl peroxide compared to wild-type B. turicatae . Finally, btpA mutants were fully infectious in a murine relapsing fever (RF) infection model. These results indicate that BtpA is either not required for mammalian infection, or that compensatory mechanisms exist in TBRF spirochetes to combat environmental stresses encountered during mammalian infection in the absence of BtpA.

The Diadenylate Cyclase CdaA Is Critical for Borrelia turicatae Virulence and Physiology

“…Because the l imit o f d etection (LOD) with our qPCR is 10 4 bacteria/ml of blood, it was possible that BtΔ cdaA bacteria are still infectious but failed to reach detectable levels ( 89 ). Therefore, we also attempted to culture bacteria out of the bloodstream of infected mice during this experiment, which is predicted to provide an LOD of 4 × 10 2 live bacteria/ml (see Materials and Methods).…”

“…c-di-AMP-dependent signaling is important for resistance to oxidative stresses in other bacterial systems (41,(51)(52)(53). While we were unable to assess the role of CdaA in resistance to oxidative stresses using media lacking pyruvate, no significant growth defect was observed in the cdaA mutant in complete mBSK medium supplemented with oxidizing agents using MIC plating assays or growth curve on April 22, 2021 by guest http://iai.asm.org/ Downloaded from analyses (data not shown) (89,109,110,115). Another phenotype associated with deletion of c-di-AMP signaling pathway components in other bacteria is altered resistance to β-lactam antibiotics, presumably due to altered cell wall metabolism (38,(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50).…”

“…Therefore, experiments were performed to assess if mutation of cdaA in B. turicatae led to altered susceptibility to oxidative stresses. Experiments to measure the sensitivity of Borrelia spirochetes to ROS are typically performed using media lacking pyruvate, as pyruvate can act as an ROS scavenger and decrease the assay sensitivity (89,114,115).…”

Genetic Manipulation of Borrelia

“…A significant advance in the Borrelia field has been the development of a genetic system for the relapsing fever (RF) spirochetes (Battisti et al, 2008;Fine et al, 2011;Guyard et al, 2013;Lopez et al, 2013;Fine et al, 2014;Raffel et al, 2014;James et al, 2016;Krishnavajhala et al, 2017;James et al, 2018;Jackson-Litteken et al, 2019). The availability of genetic tools and complete genomic sequences for multiple RF species (Lescot et al, 2008;Elbir et al, 2012;Miller et al, 2013;Barbour and Campeau Miller, 2014;Barbour, 2016;Wilder et al, 2016;Elbir et al, 2017;Kuleshov et al, 2020) has permitted investigation of genetically modified RF spirochetes in their respective tick vectors or rodent hosts, and informative comparisons with other Borrelia, as detailed in Radolf and Samuels (2021).…”

“…Most recently, a method for reliable growth of B. hermsii and B. turicatae in solid medium has been developed, allowing isolation of single colonies following genetic manipulation, as well as enumeration of colony forming units from both in vitro and in vivo samples (Raffel et al, 2018). The application of reverse genetics to the RF spirochetes has opened the door to broad investigation of these tick-borne pathogens (Raffel et al, 2014;James et al, 2016James et al, , 2018Jackson-Litteken et al, 2019;Schwan et al, 2020).…”