sets of peptides were detected in supernatants from strains D445, Bbr77 and RB50, and these included peptides corresponding to T3SS substrates previously identified using RB50 (Table 2). Bsp22, which polymerizes to form an elongated needle tip complex , BopB and BopD, which form the plasma membrane translocation apparatus [14, 29, 31], BopN, a homolog of Yersinia YscN which functions H 89 as a secreted regulator , and the BteA effector were present in supernatants from wild type strains, but absent in supernatants of ΔbscN derivatives. In the course of this analysis we discovered a novel T3SS substrate encoded from a conserved hypothetical ORF (BB1639), herein named BtrA, in supernatant fractions from RB50, D445 and Bbr77 but not from their ΔbscN derivatives. Importantly, examination of complex IV secretion substrates failed to identify unique polypeptides that were not expressed by Doramapimod RB50 or did not match the RB50 protein database. The relative amounts of T3SS substrates released into culture supernatants, as assessed by SDS-PAGE and western blot analysis, also failed to correlate with relative levels of cytotoxicity (Additional file 2 Figure S1). Although
these observations did not reveal obvious differences in the T3SS secretome that could account for the hypercytotoxic phenotypes of D445 and Bbr77, it is important to consider that the activity of the bsc T3SS and its substrate specificity are regulated at multiple levels, and results obtained using broth-grown cells provide only a crude approximation of T3SS activity during infection (see Discussion). Table 2 nLC-MSMS secretome analysis Protein name NCBI accession number Sequence coverage (%) RB50 RB50ΔbscN D445 D445ΔbscN Bbr77 Bbr77ΔbscN Bsp22 gi|33568201 41 – 59 – 60 – BopN gi|33568200 24 – 29 – 24 – BopB gi|33568205 5 – 5 – 18 – BopD gi|33568204 50 – 51 – 54 – BteA gi|33568834 7 – 6 – 28 – BtrA gi|33568223 26 – 18 – 26 – Summary of nLC-MSMS data indicated as peptide coverage for indicted T3SS substrate proteins in supernatant fractions
from B. bronchiseptica strains grown to mid-log phase in Stainer-Scholte medium. Virulence of complex IV strains during respiratory infections To determine if relative levels of cytotoxicity measured however in vitro correlate with virulence in vivo, we used a murine respiratory intranasal challenge model . Groups of 4–6 week old female specific-pathogen-free C57BL/6NCr mice were intranasally see more infected with 5 x 105 CFU. At this dose, RB50 establishes nonlethal respiratory infections that generally peak around day 10 post-inoculation and are gradually cleared from the lower respiratory tract, while persisting in the nasal cavity .As shown in Figure 4A, complex IV strains segregated into two groups. The first caused lethal infections in some (D444, Bbr77) or all (D445) of the infected animals. The second group (D446, Bbr69) caused nonlethal infections similar to RB50. Figure 4 In vivo characterization of selected complex IV B.