1a–d) To increase the resolution of the IFA observations, C bur

1a–d). To increase the resolution of the IFA observations, C. burnetii-infected Vero cells were analyzed by IEM using C. burnetii IcmT-, IcmV-, and DotH-specific antibodies. IEM

analyses revealed polar localization of the gold particle-conjugated secondary antibodies to one or both poles of C. burnetii cells when using antibodies against IcmT and DotH (Fig. OSI-744 in vitro 2). Interestingly, based on the length of a C. burnetii LCV (0.5–1.0 μm), the IEM staining of IcmT and DotH indicates that the polar localization is primarily observed on LCVs using this methodology (Fig. 2), (McCaul & Williams, 1981; McCaul, 1991; Heinzen et al., 1999). Attempts to obtain conclusive IEM results for IcmV localization were unsuccessful; however, both IcmT and DotH clearly localized to the bacterial pole(s), thus supporting the IFA observations. Using Vero cell cultures infected with C. burnetii NMII for 3 weeks

allowed for ready identification of T4BSS polar localization by IEM; however, biological questions remain about the ultrastructure of the T4BSS. Does the C. burnetii T4BSS initially localize medial to the polar region(s) and then migrate laterally to the poles during the course of cellular development or does it initially nucleate at the Ribociclib purchase pole(s) and recruit other T4BSS components to the nucleation site? The IEM images show immunoreactivity at sites somewhat medial to the C. burnetii cell pole (Fig. 2b), making this a possibility. Another observation is that the T4BSS of C. burnetii may localize on one or both pole(s) of the bacterium. The bipolar localization of the T4BSS on C. burnetii cells may correlate to cells that are approaching cell division. In such a case, the bipolar localization would ensure that daughter cells are equipped with the

components for a functional T4BSS after binary fission, hence the observation of bacteria with T4BSS localized at a single pole (Figs 1 and 2). The utility of having the T4BSS localized on the pole(s) of C. burnetii cells and how this relates to the pathogen’s interactions Rutecarpine with the host cell is not clear. The observation that A. tumefaciens intimately interfaces with a host cell at the bacterial pole (Matthysse, 1987) and that this T4ASS machinery then secretes effector molecules into the host (Christie & Vogel, 2000) would indicate that direct association of a T4SS with a membrane may allow effector secretion across/into the membrane. An analogous interface between pathogen and membrane has been observed in the intravacuolar pathogen Chlamydia trachomatis, which secretes effector proteins into the host cell via a T3SS (Fields et al., 2003) and closely associates with the PV membrane during infection (Matsumoto, 1988; Hackstadt et al., 1997). An obvious and consistent interface between a pole of C. burnetii and the PV membrane has not been demonstrated. However, a study of published EM micrographs (McCaul, 1991; Coleman et al.

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