Autotransporters are outer membrane proteins that are widely distributed among gram-negative bacteria. Like other autotransporters, the
Shigella autotransporter IcsA, which is required for actin assembly during infection, is secreted at the bacterial pole. In the bacterial cytoplasm, IcsA localizes to poles and potential cell division sites independent of the cell division protein FtsZ. To identify bacterial proteins involved in the targeting of IcsA to the pole in the bacterial cytoplasm, we screened a genome-scale library of
Escherichia coli proteins tagged with green fluorescent protein (GFP) for those that displayed a localization pattern similar to that of IcsA-GFP in cells that lack functional FtsZ using a strain carrying a temperature-sensitive
ftsZ allele. For each protein that mimicked the localization of IcsA-GFP, we tested whether IcsA localization was dependent on the presence of the protein. Although these approaches did not identify a polar receptor for IcsA, the cytoplasmic chaperone DnaK both mimicked IcsA localization at elevated temperatures as a GFP fusion and was required for the localization of IcsA to the pole in the cytoplasm of
E. coli. DnaK was also required for IcsA secretion at the pole in
Shigella flexneri. The localization of DnaK-GFP to poles and potential cell division sites was dependent on elevated growth temperature and independent of the presence of IcsA or functional FtsZ; native DnaK was found to be enhanced at midcell and the poles. A second
Shigella autotransporter, SepA, also required DnaK for secretion, consistent with a role of DnaK more generally in the chaperoning of autotransporter proteins in the bacterial cytoplasm.The
Shigella outer membrane protein IcsA is unusual in that it is secreted at the bacterial old pole (
9,
13,
24). The secreted protein forms a cap at the old pole (Fig. ), where during the infection of host cells, it interacts with cellular actin cytoskeletal proteins to induce the formation of propulsive actin tails (
6,
43,
70). Actin tail formation is essential to the spread of
Shigella spp. through cell monolayers and mammalian tissues (
6,
43,
47) and is critical for
Shigella virulence (
15,
60). IcsA is a member of the autotransporter family of secreted proteins in gram-negative bacteria. Approximately 700 autotransporter proteins are predicted to be encoded within bacterial genomes that had been annotated as of 2003 (
54). All autotransporter proteins for which the site of secretion has been determined are, like IcsA, secreted at the bacterial old pole (
35).
Open in a separate windowDesign of screen for proteins that, like IcsA, localize to potential division sites independent of FtsZ. (A) Localization of IcsA on the surface of
S. flexneri. Immunofluorescence using antibody to IcsA. (B) Localization of IcsA
507-620-GFP (expressed from pBAD24-
icsA507-620::
gfp) to poles of single cells of
E. coli MC4100
leu::Tn
10 ftsZ84(Ts) grown at the permissive temperature (30°C). (C) Localization of IcsA
507-620-GFP to potential cell division sites of
E. coli MC4100
leu::Tn
10 ftsZ84(Ts) grown at the nonpermissive temperature (42°C). (D) Diagram of the strategy used to identify proteins of
E. coli that localize to potential cell division sites independent of FtsZ, displaying a localization pattern similar to that shown for IcsA in panel C. Image from DnaK-GFP localization (expressed from leaky promoter on pCA24N-
dnaK, without induction) in screen well; incomplete overlay of GFP with phase-contrast microscopy is due to the movement of cells between capturing the two images, as cells were imaged live. Size bars = 2 μm (A and B) and 5 μm (C and D). Images are representative. O/N, overnight.Several other secreted bacterial proteins are also localized to one or both cell poles; these include the
Listeria monocytogenes actin assembly protein ActA (
39), components of the chemotaxis apparatus in
Escherichia coli and
Caulobacter crescentus (
1,
46,
66), the
Legionella pneumophila and
Agrobacterium tumifaciens type IV secretion systems (
14,
40),
Pseudomonas aeruginosa type IV pili (
8), protein components of the cell cycle regulatory pathways in
C. crescentus (reviewed in reference
72), the DNA transfer apparatus in
Bacillus subtilis and
Streptomyces spp. (
26,
28), and polar flagella in
Vibrio cholerae,
Campylobacter spp.,
Helicobacter spp.,
C. crescentus, and other gram-negative bacteria. In
L. monocytogenes, the polarity of ActA is established after ActA secretion and likely depends on differential growth rates of the cell wall along the length of the bacterium (
56). In
C. crescentus, TipN serves as a polar developmental landmark (
31,
42), and RcdA provides temporal and spatial specificity in the regulated proteolysis of key factors involved in polar asymmetry (
50). Beyond these studies, relatively little is known about the molecular mechanisms that mediate the proper localization of polar bacterial proteins.Chemical or genetic blockade of cell division leads to the formation of filamentous cells without septa. In cells that have been filamented by either blocking FtsI or depleting functional FtsZ, a cytoplasmic derivative of IcsA localizes at or near potential cell division sites (
36), which represent the sites of future cell poles. IcsA also localizes to potential division sites independent of nucleoid occlusion (
36), together indicating that the positional information directing IcsA polarity is independent of these cell division proteins and chromosome positioning. The molecules that are required for the localization of IcsA to the cell pole have not been identified.One model of IcsA localization to the pole is that freely diffusing cytoplasmic IcsA recognizes and binds a protein receptor that is present at poles and future poles. Although
icsA is present only in
Shigella spp., upon heterologous expression, IcsA localizes to the poles of other
Enterobacteriaceae (
13,
58,
59), indicating that if targeting occurs via binding to a polar receptor, the receptor is likely conserved among members of this family. In addition, since IcsA localizes independently of FtsZ and FtsI, the localization of a putative polar receptor to the pole must also be independent of these cell division proteins. To find proteins that might serve as a polar receptor for IcsA, we first conducted a genome-wide screen designed to identify the subset of
E. coli proteins that localize to poles and to potential cell division sites independently of functional FtsZ. For each conserved protein that displayed this localization pattern, we then tested whether it played a role in the polar localization of IcsA. We found that, under the conditions of our screen, a green fluorescent protein (GFP) fusion to the cytoplasmic chaperone DnaK localizes to cell poles and potential cell division sites. Although DnaK is not a polar receptor for IcsA, we demonstrated that it was required for the localization of IcsA to the pole in the bacterial cytoplasm in
E. coli and for the secretion of both IcsA and a second
Shigella autotransporter, SepA, in native
Shigella flexneri, consistent with a critical role of DnaK in the chaperoning of IcsA and SepA, and perhaps autotransporter proteins more generally, in the bacterial cytoplasm.
相似文献