Host Cell Membrane Permeability Research Articles

Exotoxin S secreted by internalized Pseudomonas aeruginosa delays lytic host cell death.

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

Differential effects of cotreatment of the antibiotic rifampin with host-directed therapeutics in reducing intracellular Staphylococcus aureus infection.

BackgroundChronic infection by Staphylococcus aureus drives pathogenesis in important clinical settings, such as recurrent pulmonary infection in cystic fibrosis and relapsing infection in osteomyelitis. Treatment options for intracellular S. aureus infection are limited. Rifampin, a lipophilic antibiotic, readily penetrates host cell membranes, yet monotherapy is associated with rapid antibiotic resistance and development of severe adverse events. Antibiotic cotreatment can reduce this progression, yet efficacy diminishes as antibiotic resistance develops. ML141 and simvastatin inhibit S. aureus invasion through host-directed rather than bactericidal mechanisms.ObjectiveTo determine whether cotreatment of ML141 or of simvastatin with rifampin would enhance rifampin efficacy.MethodsAssays to assess host cell invasion, host cell viability, host cell membrane permeability, and bactericidal activity were performed using the human embryonic kidney (HEK) 293-A cell line infected with S. aureus (29213) and treated with vehicle control, simvastatin, ML141, rifampin, or cotreatment of simvastatin or ML141 with rifampin.ResultsWe found cotreatment of ML141 with rifampin reduced intracellular infection nearly 85% when compared to the no treatment control. This decrease more than doubled the average 40% reduction in response to rifampin monotherapy. In contrast, cotreatment of simvastatin with rifampin failed to improve rifampin efficacy. Also, in contrast to ML141, simvastatin increased propidium iodide (PI) positive cells, from an average of 10% in control HEK 293-A cells to nearly 20% in simvastatin-treated cells, indicating an increase in host cell membrane permeability. The simvastatin-induced increase was reversed to control levels by cotreatment of simvastatin with rifampin.ConclusionTaken together, rifampin efficacy is increased through host-directed inhibition of S. aureus invasion by ML141, while efficacy is not increased by simvastatin. Considerations regarding novel therapeutic approaches may be dependent on underlying differences in pharmacology.

The marsupial trypanosome Trypanosoma copemani is not an obligate intracellular parasite, although it adversely affects cell health

BackgroundTrypanosoma cruzi invades and replicates inside mammalian cells, which can lead to chronic Chagas disease in humans. Trypanosoma copemani infects Australian marsupials and recent investigations indicate it may be able to invade mammalian cells in vitro, similar to T. cruzi. Here, T. cruzi 10R26 strain (TcIIa) and two strains of T. copemani [genotype 1 (G1) and genotype 2 (G2)] were incubated with marsupial cells in vitro. Live-cell time-lapse and fluorescent microscopy, combined with high-resolution microscopy (transmission and scanning electron microscopy) were used to investigate surface interactions between parasites and mammalian cells.ResultsThe number of parasites invading cells was significantly higher in T. cruzi compared to either genotype of T. copemani, between which there was no significant difference. While capable of cellular invasion, T. copemani did not multiply in host cells in vitro as there was no increase in intracellular amastigotes over time and no release of new trypomastigotes from host cells, as observed in T. cruzi. Exposure of host cells to G2 trypomastigotes resulted in increased host cell membrane permeability within 24 h of infection, and host cell death/blebbing was also observed. G2 parasites also became embedded in the host cell membrane.ConclusionsTrypanosoma copemani is unlikely to have an obligate intracellular life-cycle like T. cruzi. However, T. copemani adversely affects cell health in vitro and should be investigated in vivo in infected host tissues to better understand this host-parasite relationship. Future research should focus on increasing understanding of the T. copemani life history and the genetic, physiological and ecological differences between different genotypes.

Scylla serrata reovirus p35 protein expressed in Escherichia coli cells alters membrane permeability.

To promote viral entry, replication, release, and spread to neighboring cells, many cytolytic animal viruses encode proteins responsible for modification of host cell membrane permeability and for formation of ion channels in host cell membranes. Scylla serrata reovirus (SsRV) is a major pathogen that can severely damage mud crab (S. serrata) aquaculture. Protein p35, which is encoded by segment 10 of SsRV, contains two transmembrane domains. In this study, we found that SsRV p35 can induce membrane permeability changes when expressed in Escherichia coli. SsRV p35 expressed in bacterial cells existed as monomers under reducing conditions but formed homodimers and homotrimers under non-reducing conditions. These findings demonstrate that p35 may act as a viroporin; further studies are needed to elucidate the detailed structure-function relationships of this protein.

Membrane Integration of Poliovirus 2B Viroporin

Virus infections can result in a variety of cellular injuries, and these often involve the permeabilization of host membranes by viral proteins of the viroporin family. Prototypical viroporin 2B is responsible for the alterations in host cell membrane permeability that take place in enterovirus-infected cells. 2B protein can be localized at the endoplasmic reticulum (ER) and the Golgi complex, inducing membrane remodeling and the blockade of glycoprotein trafficking. These findings suggest that 2B has the potential to integrate into the ER membrane, but specific information regarding its biogenesis and mechanism of membrane insertion is lacking. Here, we report experimental results of in vitro translation-glycosylation compatible with the translocon-mediated insertion of the 2B product into the ER membrane as a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. A similar topology was found when 2B was synthesized in cultured cells. In addition, the in vitro translation of several truncated versions of the 2B protein suggests that the two hydrophobic regions cooperate to insert into the ER-derived microsomal membranes.

A ROLE FOR PROTEASE ACTIVITY AND HOST-CELL PERMEABILITY DURING THE PROCESS OF TRYPANOSOMA CRUZI EGRESS FROM INFECTED CELLS

The mechanism by which Trypanosoma cruzi egresses from infected cells at the end of the intracellular replication cycle is not understood. This study explored the role of T. cruzi-derived proteases and host-cell membrane permeability during the parasite's egress process. Treatment with a fluoromethyl ketone, known to inhibit the parasite's major protease, significantly reduced parasite egress. In addition, in the late stages of intracellular infection, cells infected with T. cruzi showed increased permeability as evidenced by dye exclusion tests. Furthermore, parasites could be antibody stained inside host cells without chemical permeabilization of the plasma membrane. These results suggest that in advanced stages of the intracellular cycle of T. cruzi, the host cells lose membrane integrity. Previous studies in our laboratory have found that antibodies present in sera of mice chronically infected with T. cruzi (antiegressin) bind the surface of infected cells and reduce parasite egress. In agreement with these reports, western blot analysis showed that several proteins in infected cell membrane extracts reacted with antibodies from infected mouse serum. The findings reported herein might have implications in the process of T. cruzi egress, as well as in the mechanism of action of antiegressin.

Biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein

A diverse group of cytolytic animal viruses encodes small, hydrophobic proteins to modify host cell membrane permeability to ions and small molecules during their infection cycles. In this study, we show that expression of the SARS-CoV E protein in mammalian cells alters the membrane permeability of these cells. Immunofluorescent staining and cell fractionation studies demonstrate that this protein is an integral membrane protein. It is mainly localized to the ER and the Golgi apparatus. The protein can be translocated to the cell surface and is partially associated with lipid rafts. Further biochemical characterization of the protein reveals that it is posttranslationally modified by palmitoylation on all three cysteine residues. Systematic mutagenesis studies confirm that the membrane permeabilizing activity of the SARS-CoV E protein is associated with its transmembrane domain.

Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability

To promote viral entry, replication, release, and spread to neighboring cells, many cytolytic animal viruses encode proteins responsible for modification of host cell membrane permeability and for formation of ion channels in host cell membranes during their life cycles. In this study, we show that the envelope (E) protein of severe acute respiratory syndrome-associated coronavirus can induce membrane permeability changes when expressed in Escherichia coli. E protein expressed in bacterial and mammalian cells under reducing conditions existed as monomers, but formed homodimer and homotrimer under non-reducing conditions. Site-directed mutagenesis studies revealed that two cysteine residues of the E protein were essential for oligomerization, leading to induction of membrane permeability. This is the first report demonstrating that a coronavirus-encoded protein could modify membrane permeability in E. coli cells.

Mutational Analysis of Different Regions in the Coxsackievirus 2B Protein: REQUIREMENTS FOR HOMO-MULTIMERIZATION, MEMBRANE PERMEABILIZATION, SUBCELLULAR LOCALIZATION, AND VIRUS REPLICATION

The coxsackievirus 2B protein is a small hydrophobic protein (99 amino acids) that increases host cell membrane permeability, possibly by forming homo-multimers that build membrane-integral pores. Previously, we defined the functional role of the two hydrophobic regions HR1 and HR2. Here, we investigated the importance of regions outside HR1 and HR2 for multimerization, increasing membrane permeability, subcellular localization, and virus replication through analysis of linker insertion and substitution mutants. From these studies, the following conclusions could be drawn. (i) The hydrophilic region ((58)RNHDD(62)) between HR1 and HR2 is critical for multimerization and increasing membrane permeability. Substitution analysis of Asn(61) and Asn(62) demonstrated the preference for short polar side chains (Asp, Asn), residues that are often present in turns, over long polar side chains (Glu, Gln). This finding supports the idea that the hydrophilic region is involved in pore formation by facilitating a turn between HR1 and HR2 to reverse chain direction. (ii) Studies undertaken to define the downstream boundary of HR2 demonstrated that the aromatic residues Trp(80) and Trp(82), but not the positively charged residues Arg(81), Lys(84), and Lys(86) are important for increasing membrane permeability. (iii) The N terminus is not required for multimerization but does contribute to the membrane-active character of 2B. (iv) The subcellular localization of 2B does not rely on regions outside HR1 and HR2 and does not require multimerization. (v) Virus replication requires both the membrane-active character and an additional function of 2B that is not connected to this activity.

Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy.

The 2B protein of enteroviruses is the viral membrane-active protein that is responsible for the modifications in host cell membrane permeability that take place in enterovirus-infected cells. The 2B protein shows structural similarities to the group of lytic polypeptides, polypeptides that permeate membranes either by forming multimeric membrane-integral pores or, alternatively, by lying parallel to the lipid bilayer and disturbing the curvature and symmetry of the membrane. Our aim is to gain more insight into the molecular architecture of the 2B protein in vivo. In this study, the possible existence of multimers of the coxsackie B3 virus 2B protein in single living cells was explored by fluorescence resonance energy transfer (FRET) microscopy. FRET between fusion proteins 2B-ECFP and 2B-EYFP (enhanced cyan and yellow fluorescent variants of green fluorescent protein) was monitored by using spectral imaging microscopy (SPIM) and fluorescence lifetime imaging microscopy (FLIM). Both techniques revealed the occurrence of intermolecular FRET between 2B-ECFP and 2B-EYFP, providing evidence for the formation of protein 2B homomultimers. Putative models for the mode of action of the membrane-active 2B protein and the formation of membrane-integral pores by 2B multimers are discussed.

Membrane permeabilization by small hydrophobic nonstructural proteins of Japanese encephalitis virus.

Infection with Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. We observed that JEV replication rendered infected baby hamster kidney (BHK-21) cells sensitive to the translational inhibitor hygromycin B or alpha-sarcine, to which mock-infected cells were insensitive. However, little is known about whether any JEV nonstructural (NS) proteins contribute to virus-induced changes in membrane permeability. Using an inducible Escherichia coli system, we investigated which parts of JEV NS1 to NS4 are capable of modifying membrane penetrability. We found that overexpression of NS2B-NS3, the JEV protease, permeabilized bacterial cells to hygromycin B whereas NS1 expression failed to do so. When expressed separately, NS2B alone, but not NS3, was sufficient to alter bacterial membrane permeability. Similarly, expression of NS4A or NS4B also rendered bacteria susceptible to hygromycin B inhibition. Examination of the effect of NS1 to NS4 expression on bacterial growth rate showed that NS2B exhibited the greatest inhibitory capability, followed by a modest repression from NS2A and NS4A, whereas NS1, NS3, and NS4B had only trivial influence with respect to the vector control. Furthermore, when cotransfected with a reporter gene luciferase or beta-galactosidase, transient expression of NS2A, NS2B, and NS4B markedly reduced the reporter activity in BHK-21 cells. Together, our results suggest that upon JEV infection, these four small hydrophobic NS proteins have various modification effects on host cell membrane permeability, thereby contributing in part to virus-induced cytopathic effects in infected cells.

Low pH-induced pore formation by spike proteins of enveloped viruses.

Exposure of Aedes albopictus cells infected with Semliki Forest virus (SFV; Togaviridae) to mildly acidic pH (5.6) results in a dramatic increase in the host cell membrane permeability due to pore formation by the virus spike proteins. Identical results were obtained when the cells were infected with two other viruses, Sindbis virus (SIN, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae). This permeability change could also be observed on isolated virions of SFV, SIN and VSV by measuring the influx of propidium iodide, a nucleic acid-specific fluorescent marker, into the virions. This influx was dependent on the presence of the ectodomains of the viral spikes and could be hampered by zinc ions. Furthermore, haemagglutinin, a membrane protein of influenza A virus (Orthomyxoviridae), expressed in Aedes cells induced a change in membrane permeability identical to that induced by the spike proteins of SFV, SIN and VSV when exposed to low pH. Thus acid-induced membrane permeability changes produced by spike proteins of three different virus families could be demonstrated in infected cells as well as in virions. Therefore, the low pH-induced pore formation by viral spike proteins seems to be more than an event specific for togaviruses and might well be an inherent property of enveloped viruses that use the endocytotic pathway to infect a cell.

Expression of nonstructural protein NS3 of African horsesickness virus (AHSV): evidence for a cytotoxic effect of NS3 in insect cells, and characterization of the gene products in AHSV infected Vero cells.

The smallest genome segment of African horsesickness virus (AHSV), segment 10 (S10), encodes two minor nonstructural proteins, NS3 and NS3A. While the cognate bluetongue virus (BTV) proteins have been suggested to play a role in the release of virus particles from infected cells, no function has yet been ascribed to AHSV NS3/NS3A. When the AHSV-3 S10 gene was expressed in a baculovirus system only a single NS3 protein (24 K) was synthesized, at lower levels than expected. It was shown that this could be due to a membrane association of NS3, leading to an alteration in host cell membrane permeability and eventual cell death. Based on computer predictions a general model for the membrane-associated topology of NS3 of five different orbiviruses was proposed. Studies on AHSV-3 infected Vero cells showed that equimolar amounts of NS3 and NS3A were synthesized. No evidence was found for the glycosylation of NS3. The S10 genes and NS3/3A proteins of AHSV-3 and AHSV-7 were shown to be closely related, and clearly distinct from the cognate proteins of the other 7 AHSV serotypes. This distinguishes the AHSV S10 gene product from that of BTV NS3, which appears to be much more conserved.

VIRALLY INDUCED MEMBRANE PERMEABILITY CHANGES AS A POSSIBLE LINK BETWEEN INFLUENZA B AND REYE'S SYNDROME

The mechanism by which viral infection can lead to the multiple metabolic derangements characteristic of Reye's Syndrome is not understood. The possibility that influenza virus could induce changes in membrane permeability to nutrients ordinarily concentrated within the cell was examined. Madin Darbin canine kidney (MDCK) cells were infected by egg grown influenza B virus by two routes: at physiological conditions, pH 7.4, 37°C; and adsorption at 0°C followed by brief exposure to pH 5.0, 37°C. Control cells were mock-infected with allantoic fluid. Transport (uptake and release) of phosphate (Pi) 2-deoxyglucose (dGlc) and α-aminoisobutyric acid (AIB) were measured at various intervals 0 to 10 hours after infection. At physiological pH, Pi uptake by infected cells was inhibited (p< 0.01) at 0 to 2 hours as compared to controls. Uptake of AIB was also inhibited (p< 0.01) at 0 to 2 hours. The uptake of all nutrients was higher at 6 to 10 hours in infected cells as compared to controls (p< 0.01). When release of Pi and dGlc was measured, there was no difference between infected cells and controls at 0-10 hours. At low pH, fusion occurred in infected cells, but not in control cells. Fused infected cells demonstrated both significantly increased release and diminished uptake of nutrients compared to controls. Thus influenza B virus can induce changes in host cell membrane permeability that may have important implications for cell metabolism.

Role of S gene of bacteriophage lambda in host lysis

Using E. coli lysogens carrying heat inducible λ defective in R or S or both R and S genes (called R, S and RS cells respectively), it has been found that induced β-galactosidase synthesis by host continued in S cells beyond the normal time of lysis. S function affected the permeability of host cell membrane. When radioactive labelled host membrane was incubated in vitro with extracts prepared from phage induced R and RS cells, R cell extract solubilized labelled lipid mostly in the undegraded form. This indicated that S gene product may be a lipophilic protein rather than a phospholipase.

Development of bacterial populationsin vivoin relation to plant pathogenicity

Abstract Rates of multiplication and population levels at stationary phase were determined quantitatively for pathogenic pseudomonads and non-pathogenic bacteria in leaves of Phaseolus vulgaris. Multiplication of the homologous pathogen Pseudomonas phaseolicola suggested that inhibitory factors did not regulate its behaviour. Heterologous pathogens multiplied at lower rates and to lower stationary phase population levels, and non-pathogens remained in stasis or declined slowly. A stimulation of bacteria normally limited in growth occurred when they were coinoculated with multiplying bacteria. The intercellular environment of plant tissue favoured general bacterial growth after inoculation with a multiplying organism, and it is concluded that this effect is connected with increased host cell-membrane permeability allowing leakage of cellular components which support bacterial growth.