Question the questions on CPAF Free

This is in response to the editorial commentary on CPAF substrates and secretion authored by Bavoil & Byrne (2014).

Chlamydial protease like activity factor (CPAF) was first purified in two fragments (Zhong et al., 2001) that formed intramolecular heterodimers (Dong et al., 2004a, b) for acquiring a serine protease activity (Huang et al., 2008; Chen et al., 2009, 2010a). The crystal structure revealed a proximity dependent super dimerization and sequential processing mechanism for CPAF activation (Huang et al., 2008). CPAF was characterized as a sec dependent secretion protein (Chen et al., 2010b) that localized to the host cytosol (Zhong et al., 2001; Fan et al., 2002; Heuer et al., 2003; Kleba & Stephens, 2008; Wang et al., 2011a; Bauler & Hackstadt, 2014) but without colocalization with the secreted Pgp3 protein (Li et al., 2008; Chen et al., 2010c) and implicated functionally in the interaction of a number of host cell substrate targets important to chlamydial pathogenesis (Zhong, 2011). However, in 2012, it was unambiguously shown that many of CPAF's previously identified host substrates were the result of an imprecise method that technically failed to inactivate the protease (Chen et al., 2012). This report triggered controversy and discussions on the authenticity of CPAF's function (Conrad et al., 2013; Hacker, 2014). The recent generation of CPAF deficient chlamydial organisms has provided a genetic basis for investigating the functionality of CPAF (Snavely et al., 2014). This study, based on a host cytoskeletal protein target that was affected only very late in the chlamydial growth cycle in some infected cells with lysis of the inclusion membrane, raised the question on whether CPAF is a bona fide secreted protein. Based primarily on this observation, Bavoil and Byrne took the liberty to write the aforementioned commentary that further raised the question on the authenticity of CPAF secretion. They claim that the only experimental evidence supporting CPAF secretion into the cytosol is by immunofluorescent staining that required sample processing and fixation conditions that could result in a passive leaking of CPAF into host cell cytosol. This conclusion, in the absence of any experimental findings by the authors, is too premature and academically risky as it may cause unmerited confusion among investigators; especially those who are new to the chlamydial research field and are most impressionable by the Bavoil and Byrne commentary. CPAF is a conserved protease common to all Chlamydiaceae (Dong et al., 2005) whose secretion and cytosolic location needs further investigation to definitively elucidate its function in chlamydial pathogenesis. The optimistic viewpoint of this rebuttal is that with the current advances in chlamydial genetics (Wang et al., 2011b; Ding et al., 2013; Gong et al., 2013; Johnson & Fisher, 2013; Song et al., 2013; Liu et al., 2014) we now have tools to conduct experiments that will define CPAF's function, not simply speculate on it, and will resolve this question unequivocally.

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