Tyrosine phosphorylation of proteins is an important posttranslational regulator of activity. Src-family kinases comprise a prominent family of protein tyrosine kinases that play a central role in multiple signaling pathways that regulate a diverse array of cellular activities, including adhesion, migration, differentiation, cell cycle progression, apoptosis, transcription, and intracellular trafficking (17
). The intimate relationship of intracellular pathogens with eukaryotic host cells necessitates that they adapt to and manipulate host cellular processes. Accordingly, intracellular pathogens subvert cellular signaling pathways at several stages of infection to promote entry, replication, or release during their life cycles. Src-family kinases have been shown to play important roles in the pathogenesis of a wide range of intracellular pathogens, including viruses, parasites, and bacteria (22
interacts with Src-family kinases at multiple stages during its intracellular development. The C. trachomatis
type III effector Tarp, which is secreted upon attachment and implicated in the entry process due to its actin-nucleating activity, is tyrosine phosphorylated by Src-family or other tyrosine kinases upon translocation into the host cytosol (19
). Although the role(s) of Tarp remains to be fully elucidated, the functions associated with it are independent of chlamydial transcription or translation (34
) and thus can be readily distinguished from events that are dependent upon chlamydial protein synthesis, such as trafficking to the MTOC (14
), fusion with Golgi apparatus-derived vesicles (13
), differentiation of chlamydial developmental forms (35
), and activation of Src-family kinases.
and C. pneumoniae
also appear to have evolved a dependency on Src-family kinases for intracellular trafficking and development. Recently, microdomains on the C. trachomatis
inclusion membrane were identified and shown to be comprised of at least four inclusion membrane proteins as well as activated Src-family kinases. These microdomains are thought to participate in the interactions of the chlamydial inclusion with the microtubule motor complex and function in the trafficking of the nascent inclusion to the MTOC (16
). In SYF cells or cells in which Src-family kinases are inhibited, nascent C. trachomatis
L2 inclusions are not trafficked to the MTOC and show a reduction in inclusion development with a corresponding decrease in infectious progeny. The two defects induced by a Src-family kinase deficiency appear to be unrelated, since the microtubule inhibitor nocodazole effectively blocks trafficking of nascent C. trachomatis
inclusions to the MTOC without negatively affecting inclusion development or production of infectious progeny. Nascent inclusion trafficking is dependent upon microtubules and dynein (14
). Src-family kinases have been shown to phosphorylate tubulin and bind to dynein-associated proteins (36
), suggesting that recruitment of Src-family kinases by chlamydiae may directly or indirectly mediate linkage of the inclusion to the microtubule network of the host cell. Those species examined that do not display active Src-family kinase-enriched microdomains on the inclusion membrane, C. muridarum
and C. caviae
, do not exhibit microtubule-dependent trafficking to or association with the MTOC. Whether Src-family kinase activity is required for processivity of the dynein motor complex or whether tyrosine phosphorylation contributes to the recruitment of necessary components remains to be determined. An improved understanding of C. trachomatis
trafficking may help to delineate the role of Src-family kinases in dynein motor function and microtubule trafficking.
Src-family kinases also appear to serve a separate function later in infection that is essential for normal C. trachomatis inclusion development. The observation that those few EBs which differentiate and commit to development do so normally suggests that this critical initial step of differentiation may be a stochastic process that does not favor C. trachomatis development in the absence of Src-family kinases. This developmental defect is not observed in SYF cells infected with C. caviae or C. muridarum. Indeed, C. caviae and C. muridarum replicate more efficiently in SYF cells. Although the mechanism of growth enhancement of C. caviae or C. muridarum in cells deficient in Src-family kinases is unknown, one hypothesis might be that Src-family kinases play a role in a pathway that restricts growth of these pathogens and that the absence of this pathway in SYF cells permits unregulated, enhanced growth.
Chlamydial species are well known for their distinct tissue tropisms and species specificity, despite a high degree of synteny of their genomes (39
). Few loci have been associated with disease or tissue tropism (40
). Divergence occurs primarily within a hypervariable replication termination region or plasticity zone (45
). A key distinction between human and murine chlamydial species involves their adaptations to gamma interferon (IFN-γ) (46
). In murine cell lines and in vivo
, C. muridarum
is resistant to the effects of IFN-γ, whereas C. trachomatis
is inhibited. IFN-γ-mediated inhibition of C. trachomatis
in murine cells has been attributed to the presence of p47 or immunity-related GTPases (IRGs), which are present in mice but largely absent from human cells (47
). Resistance of C. muridarum
to IFN-γ is thought to be related to the inactivation of specific IRGs by the cytotoxin that is absent or truncated in C. trachomatis
). However, treatment of a human cell line with a Src-family kinase inhibitor led to a decrease in C. trachomatis
replication similar to that observed in murine cells; thus, the mechanisms of the Src-family kinase appear to be acting though pathways other than mouse-specific IRGs. IFN-γ treatment was not investigated here; however, the plasticity zone remains a highly plausible locus for chlamydial genes responsible for the divergent responses to Src-family kinase depletion.
Early studies of tyrosine phosphorylation during chlamydial infection described two or more proteins that were tyrosine phosphorylated very early in infection and located in close apposition to the invading EBs (50
). Although the proteins were not identified, they were presumed to be of host origin. To date, ezrin has been the only host protein identified that is tyrosine phosphorylated during chlamydial invasion (52
). A more complete accounting of host and chlamydial proteins that are tyrosine phosphorylated throughout the developmental cycle will undoubtedly refine future studies designed to understand the mechanisms of growth inhibition of C. trachomatis
and growth enhancement of C. caviae
and C. muridarum
by depletion of Src-family kinases.
Protein tyrosine kinases are activated and functional at multiple stages of C. trachomatis
intracellular development. By all parameters analyzed, C. caviae
and C. muridarum
showed no activation of, recruitment of, or requirement for Src-family kinases and thus have apparently evolved life-styles that circumvent the need for Src-family kinases. Src-family kinases therefore play an important, but species-specific, role in the chlamydial developmental cycle. It is apparent that different combinations of requirements occur between species. For example, C. pneumoniae
Tarp is not phosphorylated (32
), and yet its inclusions contain tyrosine-phosphorylated microdomains (16
) and are trafficked to the MTOC. It is likely that additional functions regulated by Src-family kinases will be identified in chlamydia-infected cells. A more complete understanding of host and chlamydial tyrosine-phosphorylated proteins and the pathways associated with them should help elucidate significant questions in chlamydial biology and pathogenesis.