Outer membrane protein A (OmpA): a new player in shigella flexneri protrusion formation and inter-cellular spreading

Abstract

Outer membrane protein A (OmpA) is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92) and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the findings presented in this study.

Figure 1. Western blot analysis of OmpA expression.

OmpA expression by the wild-type S. flexneri 5a strain M90T (WT), the ompA mutant (ompA), the ompA mutant complemented with plasmid pOA (ompA/pOA) and the ompA mutant complemented with the empty vector pACYC184 (ompA/pv) is shown.WCE, whole cell extract; M, crude membrane fraction; C, cytosolic and periplasmic fraction. Fractions were suspended in 1×Laemmli buffer and heated at 37°C or at 100°C for 10 min before SDS-PAGE to evaluate the unfolded (U) and folded (F) forms of OmpA , . PVDF filters were probed using an anti-OmpA polyclonal antiserum. The positions of U and F forms of OmpA are indicated by arrows at the right side. Size markers (kDa) are shown at the left side.

Figure 2. The ompA mutation did not affect either production or secretion of IpaABCD proteins.

Proteins present in WCEs and concentrated culture supernatants (SN) of the wild-type strain (WT) and of the ompA mutant (ompA), following exposure of bacteria to 7 µg ml⁻¹ of Congo Red (CR), were stained with Comassie brilliant blue (A) or transferred to a PVDF membrane and probed using anti-IpaB and anti-IpaC antibodies (B). The positions and names of the various proteins are indicated at the right side. Size markers (kDa) are shown at the left side.

Figure 3. The ompA mutant retained wild-type invasion efficiency.

Semi-confluent HeLa cell monolayers were infected with the wild-type strain (WT), the ompA mutant (ompA), and the ompA mutant complemented with plasmid pOA (ompA/pOA), at a MOI of 20. Monolayers were fixed with methanol and Giemsa-stained for microscopic evaluation at 1 h and 3 h post-infection (insets). The icsA mutant SC560 strain was included as control (icsA). Representative images of at least four independent experiments are shown. Scale bar, 10 µm.

Figure 4. The ompA mutant retained the ability to form wild-type F-actin comet tails.

Semi-confluent HeLa cell monolayers were infected with the wild-type strain (WT), the ompA mutant (ompA), and the ompA mutant complemented with plasmid pOA (ompA/pOA). Actin was stained with rhodamine-conjugated phalloidin (red). Nuclei and bacterial DNA were stained with DAPI (blue). Representative images of double immunofluorescent microscopy are shown. Images are representatives of at least three independent experiments. Images were captured using a Leica camera and processed using Qwin software (Leica). Arrows point representative bacteria exhibiting normal F-actin comet tail formation. Scale bar, 10 µm.

Figure 5. The S. flexneri ompA mutant failed to plaque on HeLa cell monolayers.

Confluent HeLa cell monolayers were infected with the wild-type strain (WT), the ompA mutant (ompA), and the ompA mutant complemented with plasmid pOA (ompA/pOA), at a MOI of 4. The icsA mutant SC560 strain (icsA) was included as a plaque negative control. Cells were Giemsa-stained and photographed 72 h post-infection. Images are representative of at least three independent experiments.

Figure 6. LPS profiles of the wild-type strain (WT) and of the ompA mutant (ompA).

LPS was extracted as described in Materials and Methods, resolved by SDS-13%PAGE and silver stained.

Figure 7. The lack of OmpA altered exposition of IcsA at S. flexneri lateral bacterial surface.

Exponentially-growing wild-type strain (WT), the ompA mutant (ompA), and the ompA mutant complemented with plasmid pOA (ompA/pOA) were analyzed by indirect immunofluorescence using an anti-IcsA polyclonal antiserum. Representative images from at least three independent experiments are shown. Scale bar, 5 µm.

Figure 8. The lack of OmpA influenced IcsA expression.

Western blot analysis of WCE (A) and supernatants (B) of exponentially-growing wild-type strain (WT), the ompA mutant strain HND92 (ompA), the pOA-complemented derivative strain (ompA/pOA) and of the icsA mutant strain SC560 (icsA). Blots were probed with an anti-IcsA antiserum. The positions of full length mature IcsA (IcsA) of IcsP-cleaved fragment (IcsA*) and of cell associated IcsA* (ca-IcsA*), are indicated by arrows at the left side. Quantitative surface immunodetection of IcsA: (C) dot blots of intact bacteria probed with anti-IcsA antibody; and (D) intact bacteria were treated with anti-IcsA and anti-S. flexneri antibodies. The amount of antibody bound to the bacterial surface was determined by labeling with HRP-conjugated secondary antibody and measuring the HRP enzymatic activity (OD₃₇₀). Means and standard deviation of three independent experiments are shown. Blots are representative. Size markers (kDa) are shown at the right side.