A Screen for Antibiotic Resistance Determinants Reveals a Fitness Cost of the Flagellum in Pseudomonas aeruginosa.

The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa's outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic.IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.

Pseudomonas aeruginosa ExoT acts in vivo as a GTPase-activating protein for RhoA, Rac1, and Cdc42.

The Pseudomonas aeruginosa protein ExoT is a bacterial GTPase-activating protein (GAP) that has in vitro activity toward Rho, Rac, and Cdc42 GTPases. Expression of ExoT both inhibits the internalization of strain PA103 by macrophages and epithelial cells and is associated with morphological changes (cell rounding and detachment) of infected cells. We find that expression of ExoT leads to the loss of GTP-bound RhoA, Rac1, and Cdc42 in transfected HeLa cells, demonstrating that ExoT has GAP activity in vivo toward all three GTPases. GAP activity is absolutely dependent on the presence of arginine at position 149 but is not affected by whether ExoT is expressed in the absence or presence of other P. aeruginosa type III secreted proteins. We also demonstrate that expression of ExoT in epithelial cells is sufficient to cause stress fiber disassembly by means of ExoT's GAP activity toward RhoA.

Interaction of bacterial pathogens with polarized epithelium.

Many pathogens must surmount an epithelial cell barrier in order to establish an infection. While much has been learned about the interaction of bacterial pathogens with cultured epithelial cells, the influence of cell polarity on these events has only recently been appreciated. This review outlines bacterial-host epithelial cell interactions in the context of the distinct apical and basolateral surfaces of the polarized epithelium that lines the lumens of our organs.

Pseudomonas aeruginosa ExoT inhibits in vitro lung epithelial wound repair.

The nosocomial pathogen Pseudomonas aeruginosa causes clinical infection in the setting of pre-existing epithelial tissue damage, an association that is mirrored by the increased ability of P. aeruginosa to bind, invade and damage injured epithelial cells in vitro. In this study, we report that P. aeruginosa inhibits the process of epithelial wound repair in vitro through the type III-secreted bacterial protein ExoT, a GTPase-activating protein (GAP) for Rho family GTPases. This inhibition primarily targets cells at the edge of the wound, and causes actin cytoskeleton collapse, cell rounding and cell detachment. ExoT-dependent inhibition of wound repair is mediated through the GAP activity of this bacterial protein, as mutations in ExoT that alter the conserved arginine (R149) within the GAP domain abolish the ability of P. aeruginosa to inhibit wound closure. Because ExoT can also inhibit P. aeruginosa internalization by phagocytes and epithelial cells, this protein may contribute to the in vivo virulence of P. aeruginosa by allowing organisms both to overcome local host defences, such as an intact epithelial barrier, and to evade phagocytosis by immune effector cells.

Rho GTPase activity modulates Pseudomonas aeruginosa internalization by epithelial cells.

The Gram-negative pathogen Pseudomonas aeruginosa invades epithelial cells in vivo and in vitro. We have examined the pathway(s) by which epithelial cells internalize P. aeruginosa strain PA103 using Madin-Darby canine kidney (MDCK) cells. We have recently demonstrated that P. aeruginosa internalization occurs by an actin-dependent Toxin B-inhibited pathway which becomes downregulated as epithelial cells become polarized, suggesting that one or more of the Rho family GTPases is involved in bacterial internalization. Here, we demonstrate that activation of the Rho family GTPases by cytotoxic necrotizing factor 1 (CNF-1) stimulates P. aeruginosa internalization. Examination of the roles of the individual Rho family GTPases in internalization shows that expression of a constitutively active allele of RhoA (RhoAV14), but not of constitutively active Rac1 (Rac1V12) or Cdc42 (Cdc42V12), is sufficient to increase uptake of PA103pscJ. This relative increase persists when bacterial infection is established at the basolateral surface of polarized cells, suggesting that the effect of RhoAV14 is not simply due to its known ability to disrupt tight junction integrity in polarized cells. RhoAV14-mediated stimulation of bacterial uptake is actin dependent as it is abrogated by exposure to latrunculin A. We also find that endogenous Rho GTP levels in epithelial cells are increased by infection with an internalized strain of P. aeruginosa; conversely, a poorly internalized isogenic strain expressing the bacterial anti-internalization protein ExoT causes decreased Rho GTP levels. Experimental inhibition of Rho, either by expressing dominant negative RhoAN19 or by inhibiting native Rho using a membrane permeable fusion construct of a Rho-specific inhibitor, C3 ADP-ribosyltransferase, does not inhibit PA103pscJ internalization in MDCK or HeLa cells. Models consistent with these data are presented.

pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer.

Filamentous phage pIV is an outer membrane protein required for phage assembly and secretion. Chemical cross-linking and sedimentation experiments have been used to demonstrate that pIV from f1-infected Escherichia coli exists as a homo-multimer, probably composed of 10 to 12 subunits. pIV secreted from spheroplasts remains soluble and does not form multimers. Synthesis of pIV from distantly related filamentous phages or from a bacterial homolog that participates in a specialized form of extra-cellular protein secretion in the same cell with pIVf1 resulted in the formation of mixed multimers. This suggests that the homologous proteins themselves form homo-multimers. These structures could form gated channels that conduct assembling phage or specific substrate proteins across the outer membrane to the extracellular milieu.

Neuromodulator-mediated phosphorylation of specific proteins in a neurotumor hybrid cell line (NCB-20).

Mouse neuroblastoma X embryonic Chinese hamster brain explant hybrid cell line (NCB-20) forms functional synapses when intracellular cyclic AMP levels are elevated for a prolonged period of time. NCB-20 cells were labeled with [32P]orthophosphate under conditions where 2-chloroadenosine gave maximum increases of 32P incorporation into tyrosine hydroxylase in nerve growth factor dibutyryl cyclic AMP-differentiated PC12 (pheochromocytoma) cells. When NCB-20 cells were exposed to activators [5-hydroxytryptamine (5-HT), prostaglandin E1, or forskolin], resulting in activation of cyclic AMP-dependent protein kinase, increased 32P incorporation into two major proteins [130 kilodaltons (kDa) and 90 kDa] occurred. 5-HT (in the presence of phosphodiesterase inhibitor, isobutylmethylxanthine) gave a three- to fourfold increase, and forskolin a four- to sevenfold increase in 32P incorporation into the 90-kDa protein. [D-Ala2,D-Leu5]-enkephalin, which decreased cyclic AMP levels and reversed the 2-chloroadenosine-stimulated phosphorylation of tyrosine hydroxylase in differentiated PC12 cells, also reversed the stimulation of phosphorylation of the 90-kDa protein in NCB-20 cells. Pretreatment of NCB-20 cells with a calcium ionophore, A23187, gave increased phosphorylation of the 90- and 130-kDa proteins, but phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (tumor promoting agent), cell depolarization with high K+, or pretreatment with dibutyryl cyclic GMP had no effect on phosphorylation of these proteins. In contrast, phosphorylation of an 80-kDa protein was decreased by forskolin, but increased following activation of the calcium/phospholipid-dependent kinase with tumor promoting agent. Neither the 90-kDa nor the 80-kDa protein showed any immunological cross-reactivity with synapsin, a major synaptic protein known to be phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase, but not calcium/phospholipid-dependent protein kinase. This suggests that in NCB-20 cells, several unique proteins can be phosphorylated by cyclic AMP-dependent protein kinase in response to hormonal elevation of cyclic AMP levels. In contrast, an 80-kDa protein is the primary substrate for calcium/phospholipid-dependent protein kinase, and its phosphorylation is inhibited by agents that elevate cyclic AMP levels and thereby activate cyclic AMP-dependent protein kinase.