Joan C. Olson

Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane

Pseudomonas aeruginosa, an important human pathogen, preferentially binds and enters injured cells from the basolateral (BL) surface. We previously demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt are necessary and sufficient for P. aeruginosa entry from the apical (AP) surface and that AP addition of phosphatidylinositol 3,4,5-trisphosphate (PIP3) is sufficient to convert AP into BL membrane (Kierbel, A., A. Gassama-Diagne, K. Mostov, and J.N. Engel. 2005. Mol. Biol. Cell. 16:2577–2585; Gassama-Diagne, A., W. Yu, M. ter Beest, F. Martin-Belmonte, A. Kierbel, J. Engel, and K. Mostov. 2006. Nat. Cell Biol. 8:963–970). We now show that P. aeruginosa subverts this pathway to gain entry from the AP surface. In polarized monolayers, P. aeruginosa binds near cell–cell junctions without compromising them where it activates and recruits PI3K to the AP surface. Membrane protrusions enriched for PIP3 and actin accumulate at the AP surface at the site of bacterial binding. These protrusions lack AP membrane markers and are comprised of BL membrane constituents, which are trafficked there by transcytosis. The end result is that this bacterium transforms AP into BL membrane, creating a local microenvironment that facilitates its colonization and entry into the mucosal barrier.

Bacterial Biofilm Diversity in Contact Lens-Related Disease: Emerging Role of Achromobacter, Stenotrophomonas, and Delftia

PURPOSE Multi-species biofilms associated with contact lens cases and lenses can predispose individuals to contact lens-related inflammatory complications. Our study used culture-independent methods to assess the relationship between the severity of contact lens-related disease and bacteria residing in biofilms of contact lens cases and lenses. METHODS Contact lens cases and lenses from 28 patients referred to the West Virginia University Eye Institute and diagnosed as having mild keratitis, keratitis with focal infiltrates, or corneal ulcers were processed and evaluated for bacterial composition based on 16S ribosomal RNA gene sequencing. Cases and lenses from nine asymptomatic contact lens wearers were processed in a manner similar to controls. Relationships between disease severity, bacterial types, and bacterial diversity were evaluated statistically. RESULTS Disease severity and presenting visual acuity correlated with an increase in the diversity of bacterial types isolated from contact lens cases. A significant difference also was observed in the number of bacterial types associated with the three clinical groups. Achromobacter, Stenotrophomonas, and Delftia were prevalent in all disease groups, and Achromobacter and Stenotrophomonas were present in one asymptomatic control. Scanning electron microscopy revealed that Achromobacter and Stenotrophomonas formed a biofilm on the surface of contact lenses. CONCLUSIONS Culture-independent methods identified an association between disease severity and bacterial diversity in biofilms isolated from cases and lenses of patients with contact lens-related corneal disease. Achromobacter, Stenotrophomonas, and Delftia were predominant bacteria identified in our study, drawing attention to their emerging role in contact lens-related disease.

Characterization of Pseudomonas aeruginosa Exoenzyme S as a Bifunctional Enzyme in J774A.1 Macrophages

ABSTRACT Pseudomonas aeruginosa exoenzyme S (ExoS) is a type III secretion (TTS) effector, which includes both a GTPase-activating protein (GAP) activity toward the Rho family of low-molecular-weight G (LMWG) proteins and an ADP-ribosyltransferase (ADPRT) activity that targets LMWG proteins in the Ras, Rab, and Rho families. The coordinate function of both activities of ExoS in J774A.1 macrophages was assessed by using P. aeruginosa strains expressing and translocating wild-type ExoS or ExoS defective in GAP and/or ADPRT activity. Distinct and coordinated functions were identified for both domains. The GAP activity was required for the antiphagocytic effect of ExoS and was linked to interference of lamellopodium and membrane ruffle formation. Alternatively, the ADPRT activity of ExoS altered cellular adherence and morphology and was linked to effects on filopodium formation. The cellular mechanism of ExoS GAP activity included an inactivation of Rac1 function, as determined in p21-activated kinase 1-glutathione S-transferase (GST) pull-down assays. The ADPRT activity of ExoS targeted Ras and RalA but not Rab or Rho proteins, and Ral binding protein 1-GST pull-down assays identified an effect of ExoS ADPRT activity on RalA activation. The results from these studies confirm the bifunctional nature of ExoS activity within macrophages when translocated by TTS.

Use of 16S ribosomal RNA gene analyses to characterize the bacterial signature associated with poor oral health in West Virginia.

BackgroundWest Virginia has the worst oral health in the United States, but the reasons for this are unclear. This pilot study explored the etiology of this disparity using culture-independent analyses to identify bacterial species associated with oral disease.MethodsBacteria in subgingival plaque samples from twelve participants in two independent West Virginia dental-related studies were characterized using 16S rRNA gene sequencing and Human Oral Microbe Identification Microarray (HOMIM) analysis. Unifrac analysis was used to characterize phylogenetic differences between bacterial communities obtained from plaque of participants with low or high oral disease, which was further evaluated using clustering and Principal Coordinate Analysis.ResultsStatistically different bacterial signatures (P < 0.001) were identified in subgingival plaque of individuals with low or high oral disease in West Virginia based on 16S rRNA gene sequencing. Low disease contained a high frequency of Veillonella and Streptococcus, with a moderate number of Capnocytophaga. High disease exhibited substantially increased bacterial diversity and included a large proportion of Clostridiales cluster bacteria (Selenomonas, Eubacterium, Dialister). Phylogenetic trees constructed using 16S rRNA gene sequencing revealed that Clostridiales were repeated colonizers in plaque associated with high oral disease, providing evidence that the oral environment is somehow influencing the bacterial signature linked to disease.ConclusionsCulture-independent analyses identified an atypical bacterial signature associated with high oral disease in West Virginians and provided evidence that the oral environment influenced this signature. Both findings provide insight into the etiology of the oral disparity in West Virginia.

Examination of the Coordinate Effects of Pseudomonas aeruginosa ExoS on Rac1

ABSTRACT Exoenzyme S (ExoS) is a bifunctional toxin directly translocated into eukaryotic cells by the Pseudomonas aeruginosa type III secretory (TTS) process. The amino-terminal GTPase-activating (GAP) activity and the carboxy-terminal ADP-ribosyltransferase (ADPRT) activity of ExoS have been found to target but exert opposite effects on the same low-molecular-weight G protein, Rac1. ExoS ADP-ribosylation of Rac1 is cell line dependent. In HT-29 human epithelial cells, where Rac1 is ADP-ribosylated by TTS-ExoS, Rac1 was activated and relocalized to the membrane fraction. Arg66 and Arg68 within the GTPase-binding region of Rac1 were identified as preferred sites of ExoS ADP-ribosylation. The modification of these residues by ExoS would be predicted to interfere with Rac1 inactivation and explain the increase in active Rac1 caused by ExoS ADPRT activity. Using ExoS-GAP and ADPRT mutants to examine the coordinate effects of the two domains on Rac1 function, limited effects of ExoS-GAP on Rac1 inactivation were evident in HT-29 cells. In J774A.1 macrophages, where Rac1 was not ADP-ribosylated, ExoS caused a decrease in the levels of active Rac1, and this decrease was linked to ExoS-GAP. Using immunofluorescence staining of Rac1 to understand the cellular basis for the targeting of ExoS ADPRT activity to Rac1, an inverse relationship was observed between Rac1 plasma membrane localization and Rac1 ADP-ribosylation. The results obtained from these studies have allowed the development of a model to explain the differential targeting and coordinate effects of ExoS GAP and ADPRT activity on Rac1 within the host cell.

Role of host cell polarity and leading edge properties in Pseudomonas type III secretion.

Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration.

Characterization of an ExoS Type III Translocation-Resistant Cell Line

ABSTRACT Pseudomonas aeruginosa ExoS is a type III-secreted type III-secreted, bifunctional protein that causes diverse effects on eukaryotic cell function. The coculture of P. aeruginosa strains expressing ExoS with HL-60 myeloid cells revealed the cell line to be resistant to the toxic effects of ExoS. Differentiation of HL-60 cells with phorbol 12-myristate 13-acetate (TPA) rendered the cell line sensitive to ExoS. To understand the cellular basis for the alteration in sensitivity, undifferentiated and TPA-differentiated HL-60 cells were compared for differences in bacterial adherence, type III secretion induction, and ExoS translocation. These comparisons found that ExoS was translocated more efficiently in TPA-differentiated HL-60 cells than in undifferentiated cells. The studies support the ability of eukaryotic cells to influence P. aeruginosa TTS at the level of membrane translocation.

Regulation of Rab5 Function during Phagocytosis of Live Pseudomonas aeruginosa in Macrophages

ABSTRACT Pseudomonas aeruginosa, a Gram-negative opportunistic human pathogen, is a frequent cause of severe hospital-acquired infections. Effectors produced by the type III secretion system disrupt mammalian cell membrane trafficking and signaling and are integral to the establishment of P. aeruginosa infection. One of these effectors, ExoS, ADP-ribosylates several host cell proteins, including Ras and Rab GTPases. In this study, we demonstrated that Rab5 plays a critical role during early stages of P. aeruginosa invasion of J774-Eclone macrophages. We showed that live, but not heat-inactivated, P. aeruginosa inhibited phagocytosis and that this occurred in conjunction with downregulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and in J744-Eclone cells, ExoS ADP-ribosyltransferase activity caused a more severe inhibition of phagocytosis than ExoS Rho GTPase activity. Furthermore, we found that expression of Rin1, a Rab5 guanine exchange factor, but not Rabex5 and Rap6, partially reversed the inactivation of Rab5 during invasion of live P. aeruginosa. These studies provide evidence that live P. aeruginosa cells are able to influence their rate of phagocytosis in macrophages by directly regulating activation of Rab5.

Examining the Role of Actin-Plasma Membrane Association in Pseudomonas aeruginosa Infection and Type III Secretion Translocation in Migratory T24 Epithelial Cells

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa targets wounded epithelial barriers, but the cellular alteration that increases susceptibility to P. aeruginosa infection remains unclear. This study examined how cell migration contributes to the establishment of P. aeruginosa infections using (i) highly migratory T24 epithelial cells as a cell culture model, (ii) mutations in the type III secretion (T3S) effector ExoS to manipulate P. aeruginosa infection, and (iii) high-resolution immunofluorescent microscopy to monitor ExoS translocation. ExoS includes both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities, and P. aeruginosa cells expressing wild-type ExoS preferentially bound to the leading edge of T24 cells, where ExoS altered leading-edge architecture and actin anchoring in conjunction with interrupting T3S translocation. Inactivation of ExoS GAP activity allowed P. aeruginosa to be internalized and secrete ExoS within T24 cells, but as with wild-type ExoS, translocation was limited in association with disruption of actin anchoring. Inactivation of ExoS ADPRT activity resulted in significantly enhanced T3S translocation by P. aeruginosa cells that remained extracellular and in conjunction with maintenance of actin-plasma membrane association. Infection with P. aeruginosa expressing ExoS lacking both GAP and ADPRT activities resulted in the highest level of T3S translocation, and this occurred in conjunction with the entry and alignment of P. aeruginosa and ExoS along actin filaments. Collectively, in using ExoS mutants to modulate and visualize T3S translocation, we were able to (i) confirm effector secretion by internalized P. aeruginosa, (ii) differentiate the mechanisms underlying the effects of ExoS GAP and ADPRT activities on P. aeruginosa internalization and T3S translocation, (iii) confirm that ExoS ADPRT activity targeted a cellular substrate that interrupted T3S translocation, (iv) visualize the ability of P. aeruginosa and ExoS to align with actin filaments, and (v) demonstrate an association between actin anchoring at the leading edge of T24 cells and the establishment of P. aeruginosa infection. Our studies also highlight the contribution of ExoS to the opportunistic nature of P. aeruginosa infection through its ability to exert cytotoxic effects that interrupt T3S translocation and P. aeruginosa internalization, which in turn limit the P. aeruginosa infectious process.

Metastatic MTLn3 and non-metastatic MTC adenocarcinoma cells can be differentiated by Pseudomonas aeruginosa

Summary Cancer patients are known to be highly susceptible to Pseudomonas aeruginosa (Pa) infection, but it remains unknown whether alterations at the tumor cell level can contribute to infection. This study explored how cellular changes associated with tumor metastasis influence Pa infection using highly metastatic MTLn3 cells and non-metastatic MTC cells as cell culture models. MTLn3 cells were found to be more sensitive to Pa infection than MTC cells based on increased translocation of the type III secretion effector, ExoS, into MTLn3 cells. Subsequent studies found that higher levels of ExoS translocation into MTLn3 cells related to Pa entry and secretion of ExoS within MTLn3 cells, rather than conventional ExoS translocation by external Pa. ExoS includes both Rho GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT) enzyme activities, and differences in MTLn3 and MTC cell responsiveness to ExoS were found to relate to the targeting of ExoS-GAP activity to Rho GTPases. MTLn3 cell migration is mediated by RhoA activation at the leading edge, and inhibition of RhoA activity decreased ExoS translocation into MTLn3 cells to levels similar to those of MTC cells. The ability of Pa to be internalized and transfer ExoS more efficiently in association with Rho activation during tumor metastasis confirms that alterations in cell migration that occur in conjunction with tumor metastasis contribute to Pa infection in cancer patients. This study also raises the possibility that Pa might serve as a biological tool for dissecting or detecting cellular alterations associated with tumor metastasis.