HtrA, a Temperature- and Stationary Phase-Activated Protease Involved in Maturation of a Key Microbial Virulence Determinant, Facilitates Borrelia burgdorferi Infection in Mammalian Hosts

Abstract

d High-temperature requirement protease A (HtrA) represents a family of serine proteases that play important roles in microbial biology. Unlike the genomes of most organisms, that of Borrelia burgdorferi notably encodes a single HtrA gene product, termed BbHtrA. Previous studies identified a few substrates of BbHtrA; however, their physiological relevance could not be ascertained, as targeted deletion of the gene has not been successful. Here we show that BbhtrA transcripts are induced during spirochete growth either in the stationary phase or at elevated temperature. Successful generation of a BbhtrA deletion mutant and restoration by genetic complementation suggest a nonessential role for this protease in microbial viability; however, its remarkable growth, morphological, and structural defects during cultivation at 37°C confirm a high-temperature requirement for protease activation and function. The BbhtrA-deficient spirochetes were unable to establish infection of mice, as evidenced by assessment of culture, PCR, and serology. We show that transcript abundance as well as proteolytic processing of a borrelial protein required for cell fission and infectivity, BB0323, is impaired in BbhtrA mutants grown at 37°C, which likely contributed to their inability to survive in a mammalian host. Together, these results demonstrate the physiological relevance of a unique temperature-regulated borrelial protease, BbHtrA, which further enlightens our knowledge of intriguing aspects of spirochete biology and infectivity. L yme disease is a common vector-borne infectious disease in the Northern Hemisphere, posing a serious health threat to humans and animals (1-3). Newly revised estimates from the Centers for Disease Control and Prevention suggest that there are likely to be over 300,000 new cases per year in the United States alone, and a vaccine to prevent human infection is currently unavailable. The disease can be transmitted via the bite of the infected arthropod vector, the Ixodes scapularis tick, harboring the spirochete pathogen Borrelia burgdorferi, often resulting in serious illness in susceptible hosts (2, 3). While most Lyme disease cases can be treated with an antibiotic, a minority of patients will have persistent or relapsing nonobjective symptoms that vary in intensity, are nonresponsive to further antibiotic therapy, and are collectively termed chronic Lyme disease or posttreatment Lyme disease syndrome, the underlying mechanism and pathogenesis of which remain highly controversial (4). Detection of early Lyme disease, when antibiotic treatment is most effective, also remains challenging, as the infection reflects many nonspecific symptoms that are shared by many other febrile or influenza-like diseases (5, 6). Thus, the development of efficient diagnostics, vaccines, or new effective drugs is a high-priority goal that requires a thorough understanding of the unusual biology and infection process of this pathogen.The pathogen of Lyme disease exhibits evolutionary divergence from other bacteria, even related...

Characterization of 6S RNA in the Lyme disease spirochete

“…While only vlsE mutants (Bankhead & Chaconas, ) and lmp1 mutants (Yang, Coleman, Anguita, & Pal, ), which lack surface‐exposed lipoproteins involved in evading host immunity, seroconverted in the absence of infection. An htrA mutant strain, lacking a virulence‐associated protease, elicited a weak immune response without being able to establish infection (Ye et al, ). The murine immune system reacted to considerably fewer antigenic proteins in the ssrS null mutant compared to either the wild‐type or ssrS complemented strains, which could be due to Bb6S RNA regulating expression of proteins targeted by the adaptive immune system and, possibly, involved in host interactions, but these questions await further investigation.…”

Borrelia burgdorferisurface-located Lmp1 protein processed into region-specific polypeptides that are critical for microbial persistence

“…The Lmp1 cleavage activity was absent in a catalytic BbHtrA mutant (Kariu et al ., 2013b). Second, processing of Lmp1 in spirochetes was impaired in isogenic BbHtrA-deficient mutants (Ye et al ., 2016) but was comparable in wild-type and BbHtrA-complemented isolates; notably, the difference of Lmp1 processing was not recorded in spirochetes grown at 34°C but obvious at 37°C where protease activity of BbHtrA is known to be activated (Clausen et al ., 2002, Clausen et al ., 2011, Coleman et al ., 2013, Kariu et al ., 2013b). This also suggests an artificial or nonspecific cleavage during cell lysis is unlikely as our preparations were performed at room temperature (where BbHtrA or other proteases are inefficient) and also includes the presence of protease inhibitors.…”

“…Bacteria were cultivated in Barbour-Stoenner-Kelly II (BSK-II) medium at 34°C or at 37°C. For genetic manipulation and maintenance, Lmp1- or BbHtrA-deficient spirochetes were cultivated with appropriate antibiotics (Yang et al ., 2009, Yang et al ., 2010, Yang et al ., 2016, Ye et al ., 2016). Four- to six-week-old C3H/HeN mice were purchased from the National Institutes of Health and Charles River laboratories.…”

A joint effort: The interplay between the innate and the adaptive immune system in Lyme arthritis

“…High‐temperature requirement protease A is a well‐known family of adenosine triphosphate‐independent serine proteases that can break down ECM components. B. burgdorferi encodes such a protease, supporting Borrelial tissue invasion through ECM degradation …”