AUTHOR=Ficht Thomas A., Pei Jianwu , Kahl-McDonagh Melissa 

TITLE=Brucella dissociation is essential for macrophage egress and bacterial dissemination

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=4

YEAR=2014

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2014.00023

DOI=10.3389/fcimb.2014.00023

ISSN=2235-2988

ABSTRACT=<p>It has long been observed that smooth <italic>Brucella</italic> can dissociate into rough mutants that are cytotoxic to macrophages. However, the <italic>in vivo</italic> biological significance and/or mechanistic details of <italic>Brucella</italic> dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using <italic>Brucella</italic> strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while <italic>Brucella</italic> uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4–5 days post infection (p.i.) with cytotoxic <italic>Brucella</italic> 16MΔ<italic>manBA</italic> at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating <italic>Brucella</italic>. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔ<italic>manBA</italic>Δ<italic>virB2</italic> at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating <italic>Brucella</italic> in the monolayers infected with 16MΔ<italic>manBA</italic>Δ<italic>virB2</italic>. The size of the foci observed in macrophage monolayers infected with rough <italic>Brucella</italic> correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for <italic>Brucella</italic> egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that <italic>Brucella</italic>-induced cytotoxicity is critical for <italic>Brucella</italic> egress and dissemination.</p>