Genomic comparison among several B. burgdorferi sensu stricto (s.s.) strains reveals highly conserved BBF01/arp sequences (95-100% identity from GenBank Blast). Curiously, the genomes of other B. burgdorferi sensu lato strains that are available in GenBank,
such as B. afzelii and B. garinii, do not appear to have an arp homolog. In contrast to arp conservation in B. burgdorferi s.s. strains, dbpA and ospC, which also encode immunogenic antigens that are expressed during infection [19, 21–23], have considerable variation (81-85% identity) among the same B. burgdorferi s.s. strains (GenBank). As noted, both Arp and DbpA stimulate an arthritis-resolving immune response , and DbpA and OspC elicit protective immune responses against challenge [11, 14, 24]. It is therefore curious that Arp has such Crenigacestat a conserved sequence among B. burgdorferi s.s. strains, when it is so obviously subjected to immune selection pressure. The present study explored the biological behavior of B. burgdorferi devoid of, or complemented with, Arp. Arp was found to be non-essential for infectivity, but it influenced infectious dose, spirochete burdens in tissues, arthritis severity, and tick infection kinetics, underscoring its biological significance.
Results Seven B. burgdorferi B31-arp deletion mutants (Δarp) were created, and found to grow equally well in BSKII medium as B31 (wild-type) spirochetes. The 7 Δarp mutants were initially tested for infectivity in infant ICR mice, which serve as an inexpensive system for titrating infectivity . All seven mutants were determined to be flagellin B (flaB) DNA-positive and arp DNA-negative buy AZD1480 by polymerase chain reaction (PCR), following growth selection in streptomycin. Four 2-day-old mice were inoculated with 106 of each Δarp mutant or wild-type spirochetes,
Carnitine dehydrogenase and sub-inoculation site and PCI32765 urinary bladder were cultured to determine infectivity and ability to disseminate at 7 and 21 days after inoculation. All were infectious, and all disseminated to the urinary bladder. Spirochetes cultured from the inoculation site and urinary bladder were tested by PCR for presence of flaB and arp. Urinary bladder isolates from mice that were flaB-positive and arp-negative were selected for further analysis and confirmed to be arp-null. Upon subsequent inoculation of infant ICR mice with wild-type or each of the seven Δarp mutants, arthritis was of equivalent severity as mice infected with B31 among all groups of mice, indicating that B. burgdorferi devoid of arp were not only infectious, but also equally pathogenic as wild-type B. burgdorferi in susceptible infant mice. One arp isolate (Δarp3) was selected for further analysis. The median infectious dose (ID50) of Δarp3 was compared to wild-type and to Δarp3 complemented with the plasmid lp28-1G containing arp (Δarp3 + lp28-1G). Groups of 4 infant ICR mice were inoculated subdermally with 101, 102, 103, 104, or 105 spirochetes.