scholarly journals Putative Stress Sensors WscA and WscB Are Involved in Hypo-Osmotic and Acidic pH Stress Tolerance in Aspergillus nidulans

2011 ◽  
Vol 10 (11) ◽  
pp. 1504-1515 ◽  
Author(s):  
Taiki Futagami ◽  
Seiki Nakao ◽  
Yayoi Kido ◽  
Takuji Oka ◽  
Yasuhiro Kajiwara ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Nidulans ◽  
Signaling Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Acidic Ph ◽  
Content Type ◽  
Downstream Signaling ◽  
Soluble Cell ◽  
Stress Sensors ◽  
In The Wild

ABSTRACT Wsc proteins have been identified in fungi and are believed to be stress sensors in the cell wall integrity (CWI) signaling pathway. In this study, we characterized the sensor orthologs WscA and WscB in Aspergillus nidulans . Using hemagglutinin-tagged WscA and WscB, we showed both Wsc proteins to be N - and O -glycosylated and localized in the cell wall and membrane, implying that they are potential cell surface sensors. The wscA disruptant (Δ wscA ) strain was characterized by reduced colony and conidia formation and a high frequency of swollen hyphae under hypo-osmotic conditions. The deficient phenotype of the Δ wscA strain was facilitated by acidification, but not by alkalization or antifungal agents. In contrast, osmotic stabilization restored the normal phenotype in the Δ wscA strain. A similar inhibition was observed in the wscB disruptant strain, but to a lesser extent. In addition, a double wscA and wscB disruptant (Δ wscA Δ wscB ) strain was viable, but its growth was inhibited to a greater degree, indicating that the functions of the products of these genes are redundant. Transcription of α -1,3-glucan synthase genes ( agsA and agsB ) was significantly altered in the wscA disruptant strain, resulting in an increase in the amount of alkali-soluble cell wall glucan compared to that in the wild-type (wt) strain. An increase in mitogen-activated protein kinase (MpkA) phosphorylation was observed as a result of wsc disruption. Moreover, the transient transcriptional upregulation of the agsB gene via MpkA signaling was observed in the Δ wscA Δ wscB strain to the same degree as in the wt strain. These results indicate that A. nidulans Wsc proteins have a different sensing spectrum and downstream signaling pathway than those in the yeast Saccharomyces cerevisiae and that they play an important role in CWI under hypo-osmotic and acidic pH conditions.

scholarly journals MpkA-Dependent and -Independent Cell Wall Integrity Signaling in Aspergillus nidulans

2007 ◽  
Vol 6 (8) ◽  
pp. 1497-1510 ◽  
Author(s):  
Tomonori Fujioka ◽  
Osamu Mizutani ◽  
Kentaro Furukawa ◽  
Natsuko Sato ◽  
Akira Yoshimi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Transcriptional Regulation ◽  
Aspergillus Nidulans ◽  
Cell Wall Integrity ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Wild Type ◽  
Glucan Synthase ◽  
Cell Wall Biogenesis ◽  
In The Wild

ABSTRACT Cell wall integrity signaling (CWIS) maintains cell wall biogenesis in fungi, but only a few transcription factors (TFs) and target genes downstream of the CWIS cascade in filamentous fungi are known. Because a mitogen-activated protein kinase (MpkA) is a key CWIS enzyme, the transcriptional regulation of mpkA and of cell wall-related genes (CWGs) is important in cell wall biogenesis. We cloned Aspergillus nidulans mpkA; rlmA, a TF gene orthologous to Saccharomyces cerevisiae RLM1 that encodes Rlm1p, a major Mpk1p-dependent TF that regulates the transcription of MPK1 besides that of CWGs; and Answi4 and Answi6, homologous to S. cerevisiae SWI4 and SWI6, encoding the Mpk1p-activating TF complex Swi4p-Swi6p, which regulates CWG transcription in a cell cycle-dependent manner. A. nidulans rlmA and mpkA cDNA functionally complemented S. cerevisiae rlm1Δ and mpk1Δ mutants, respectively, but Answi4 and Answi6 cDNA did not complement swi4Δ and swi6Δ mutants. We constructed A. nidulans rlmA, Answi4 and Answi6, and mpkA disruptants (rlmAΔ, Answi4Δ Answi6Δ, and mpkAΔ strains) and analyzed mpkA and CWG transcripts after treatment with a β-1,3-glucan synthase inhibitor (micafungin) that could activate MpkA via CWIS. Levels of mpkA transcripts in the mutants as well as those in the wild type were changed after micafungin treatment. The β-glucuronidase reporter gene controlled by the mpkA promoter was expressed in the wild type but not in the mpkAΔ strain. Thus, mpkA transcription seems to be autoregulated by CWIS via MpkA but not by RlmA or AnSwi4-AnSwi6. The transcription of most CWGs except α-1,3-glucan synthase genes (agsA and agsB) was independent of RlmA and AnSwi4-AnSwi6 and seemed to be regulated by non-MpkA signaling. The transcriptional regulation of mpkA and of CWGs via CWIS in A. nidulans differs significantly from that in S. cerevisiae.

scholarly journals Aspergillus nidulans Protein O-Mannosyltransferases Play Roles in Cell Wall Integrity and Developmental Patterning

2009 ◽  
Vol 8 (10) ◽  
pp. 1475-1485 ◽  
Author(s):  
Thanyanuch Kriangkripipat ◽  
Michelle Momany
Keyword(s):  
Cell Wall ◽  
Aspergillus Nidulans ◽  
Double Mutant ◽  
Elevated Temperatures ◽  
Cell Wall Integrity ◽  
Wild Type ◽  
Spatial Cues ◽  
Developmental Patterning ◽  
In The Wild ◽  
Increased Sensitivity

ABSTRACT Protein O-mannosyltransferases (Pmts) initiate O-mannosyl glycan biosynthesis from Ser and Thr residues of target proteins. Fungal Pmts are divided into three subfamilies, Pmt1, -2, and -4. Aspergillus nidulans possesses a single representative of each Pmt subfamily, pmtA (subfamily 2), pmtB (subfamily 1), and pmtC (subfamily 4). In this work, we show that single Δpmt mutants are viable and have unique phenotypes and that the ΔpmtA ΔpmtB double mutant is the only viable double mutant. This makes A. nidulans the first fungus in which all members of individual Pmt subfamilies can be deleted without loss of viability. At elevated temperatures, all A. nidulans Δpmt mutants show cell wall-associated defects and increased sensitivity to cell wall-perturbing agents. The Δpmt mutants also show defects in developmental patterning. Germ tube emergence is early in ΔpmtA and more frequent in ΔpmtC mutants than in the wild type. In ΔpmtB mutants, intrahyphal hyphae develop. All Δpmt mutants show distinct conidiophore defects. The ΔpmtA strain has swollen vesicles and conidiogenous cells, the ΔpmtB strain has swollen conidiophore stalks, and the ΔpmtC strain has dramatically elongated conidiophore stalks. We also show that AN5660, an ortholog of Saccharomyces cerevisiae Wsc1p, is modified by PmtA and PmtC. The Δpmt phenotypes at elevated temperatures, increased sensitivity to cell wall-perturbing agents and restoration to wild-type growth with osmoticum suggest that A. nidulans Pmts modify proteins in the cell wall integrity pathway. The altered developmental patterns in Δpmt mutants suggest that A. nidulans Pmts modify proteins that serve as spatial cues.

scholarly journals Mitogen-Activated Protein Kinase Cross-Talk Interaction Modulates the Production of Melanins in Aspergillus fumigatus

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Adriana Oliveira Manfiolli ◽  
Filipe Silva Siqueira ◽  
Thaila Fernanda dos Reis ◽  
Patrick Van Dijck ◽  
Sanne Schrevens ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Cross Talk ◽  
Pathogenic Fungus ◽  
Antifungal Drugs ◽  
Mitogen Activated Protein Kinase ◽  
Melanin Production ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Salvage Pathways

ABSTRACT The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade. IMPORTANCE Aspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.

scholarly journals Cross Talk between the Cell Wall Integrity and Cyclic AMP/Protein Kinase A Pathways in Cryptococcus neoformans

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Maureen J. Donlin ◽  
Rajendra Upadhya ◽  
Kimberly J. Gerik ◽  
Woei Lam ◽  
Laura G. VanArendonk ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Cryptococcus Neoformans ◽  
Signaling Pathway ◽  
Cell Wall Integrity ◽  
Wild Type ◽  
Content Type ◽  
Kinase A

ABSTRACTCryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely,PKC1,BCK1,MKK2, andMPK1results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions ofBCK1,MKK2, andMPK1compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis.IMPORTANCECryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. neoformans, and biosynthesis and repair of the wall are primarily controlled by the cell wall integrity (CWI) signaling pathway. In this study, we demonstrate that deletion of any of three core kinases in the CWI pathway impacts not only the cell wall but also the amount of surface capsule. Deletion of any of the kinases results in significantly reduced cellular cyclic AMP (cAMP) levels, and addition of exogenous cAMP rescues the capsule defect and some cell wall defects, supporting a direct role for the CWI pathway in regulation of capsule in conjunction with the cAMP/protein kinase A pathway.

scholarly journals Cryptococcus neoformans Phosphoinositide-Dependent Kinase 1 (PDK1) Ortholog Is Required for Stress Tolerance and Survival in Murine Phagocytes

2012 ◽  
Vol 12 (1) ◽  
pp. 12-22 ◽  
Author(s):  
Yeissa Chabrier-Roselló ◽  
Kimberly J. Gerik ◽  
Kristy Koselny ◽  
Louis DiDone ◽  
Jennifer K. Lodge ◽  
...  
Keyword(s):  
Cell Wall ◽  
Stress Tolerance ◽  
Cryptococcus Neoformans ◽  
Nitrosative Stress ◽  
Galleria Mellonella ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Oxidative And Nitrosative Stress ◽  
Independent Manner ◽  
Content Type

ABSTRACTCryptococcus neoformansPKH2-01andPKH2-02are orthologous to mammalian PDK1 kinase genes. Although orthologs of these kinases have been extensively studied inS. cerevisiae, little is known about their function in pathogenic fungi. In this study, we show thatPKH2-02but notPKH2-01is required forC. neoformansto tolerate cell wall, oxidative, nitrosative, and antifungal drug stress. Deletion ofPKH2-02leads to decreased basal levels of Pkc1 activity and, consequently, reduced activation of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway in response to cell wall, oxidative, and nitrosative stress.PKH2-02function also is required for tolerance of fluconazole and amphotericin B, two important drugs for the treatment of cryptococcosis. Furthermore, OSU-03012, an inhibitor of human PDK1, is synergistic and fungicidal in combination with fluconazole. Using aGalleria mellonellamodel of low-temperature cryptococcosis, we found thatPKH2-02is also required for virulence in a temperature-independent manner. Consistent with the hypersensitivity of thepkh2-02Δ mutant to oxidative and nitrosative stress, this mutant shows decreased survival in murine phagocytes compared to that of wild-type (WT) cells. In addition, we show that deletion ofPKH2-02affects the interaction betweenC. neoformansand phagocytes by decreasing its ability to suppress production of tumor necrosis factor alpha (TNF-α) and reactive oxygen species. Taken together, our studies demonstrate that Pkh2-02-mediated signaling inC. neoformansis crucial for stress tolerance, host-pathogen interactions, and both temperature-dependent and -independent virulence.

scholarly journals Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of β-Lactamase-Producing Escherichia coli

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Kristin R. Baker ◽  
Helga Høeg Sigurðardóttir ◽  
Bimal Jana ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Lag Phase ◽  
Growth Curve Analysis ◽  
Wild Type ◽  
Content Type ◽  
Cell Wall Biogenesis ◽  
Genes Encoding ◽  
In The Wild ◽  
Nonessential Genes

ABSTRACT Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for “helper” drugs reversing cephem resistance in Escherichia coli strains producing β-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two nonessential genes involved in cell wall biogenesis potentiates cephem activity according to drug- and strain-specific patterns.

scholarly journals Role of the Guanine Nucleotide Exchange Factor Rom2 in Cell Wall Integrity Maintenance of Aspergillus fumigatus

2012 ◽  
Vol 12 (2) ◽  
pp. 288-298 ◽  
Author(s):  
Sweta Samantaray ◽  
Michael Neubauer ◽  
Christoph Helmschrott ◽  
Johannes Wagener
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Guanine Nucleotide Exchange Factor ◽  
Cell Wall Integrity ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Content Type ◽  
Guanine Nucleotide Exchange ◽  
Stress Sensors ◽  
Nucleotide Exchange Factor

ABSTRACTAspergillus fumigatusis a mold and the causal agent of invasive aspergillosis, a systemic disease with high lethality. Recently, we identified and functionally characterized three stress sensors implicated in the cell wall integrity (CWI) signaling of this pathogen, namely, Wsc1, Wsc3, and MidA. Here, we functionally characterize Rom2, a guanine nucleotide exchange factor with essential function for the cell wall integrity ofA. fumigatus. A conditionalrom2mutant has severe growth defects under repressive conditions and incorporates all phenotypes of the three cell wall integrity sensor mutants, e.g., the echinocandin sensitivity of the Δwsc1mutant and the Congo red, calcofluor white, and heat sensitivity of the ΔmidAmutant. Rom2 interacts with Rho1 and shows a similar intracellular distribution focused at the hyphal tips. Our results place Rom2 between the cell surface stress sensors Wsc1, Wsc3, MidA, and Rho1 and their downstream effector mitogen-activated protein (MAP) kinase module Bck1-Mkk2-MpkA.

scholarly journals Signaling Domains of Mucin Msb2 in Candida albicans

2015 ◽  
Vol 14 (4) ◽  
pp. 359-370 ◽  
Author(s):  
Marc Swidergall ◽  
Lasse van Wijlick ◽  
Joachim F. Ernst
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Map Kinase ◽  
Cytoplasmic Tail ◽  
Wall Stress ◽  
Mitogen Activated Protein Kinase ◽  
Basal Resistance ◽  
Interacting Protein ◽  
Content Type ◽  
Cell Wall Stress

ABSTRACTCandida albicansadapts to the human host by environmental sensing using the Msb2 signal mucin, which regulates fungal morphogenesis and resistance characteristics. Msb2 is anchored within the cytoplasmic membrane by a single transmembrane (TM) region dividing it into a large N-terminal exodomain, which is shed, and a small cytoplasmic domain. Analyses of strains carrying deleted Msb2 variants revealed an exodomain segment required for cleavage, shedding, and all functions of Msb2. Phosphorylation of the mitogen-activated protein kinase (MAP kinase) Cek1 was regulated by three distinct regions in Msb2: in unstressed cells, N-terminal sequences repressed phosphorylation, while its induction under cell wall stress required the cytoplasmic tail (C-tail) and sequences N-terminally flanking the TM region, downstream of the proposed cleavage site. Within the latter Msb2 region, overlapping but not identical sequences were also required for hyphal morphogenesis, basal resistance to antifungals, and, in unstressed cells, downregulation of thePMT1transcript, encoding proteinO-mannosyltransferase-1. Deletion of two-thirds of the exodomain generated a truncated Msb2 variant with a striking ability to induce hyperfilamentous growth, which depended on the presence of the Msb2-interacting protein Sho1, the MAP kinase Cek1, and the Efg1 transcription factor. Under cell wall stress, the cytoplasmic tail relocalized partially to the nucleus and contributed to regulation of 117 genes, as revealed by transcriptomic analyses. Genes regulated by the C-tail contained binding sites for the Ace2 and Azf1 transcription factors and included theALScell wall genes. We concluded that Msb2 fulfills its numerous functions by employing functional domains that are distributed over its entire length.

scholarly journals Abolishing Cell Wall Glycosylphosphatidylinositol-Anchored Proteins in Candida albicans Enhances Recognition by Host Dectin-1

2015 ◽  
Vol 83 (7) ◽  
pp. 2694-2704 ◽  
Author(s):  
Hui Shen ◽  
Si Min Chen ◽  
Wei Liu ◽  
Fang Zhu ◽  
Li Juan He ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fungal Infections ◽  
Nuclear Factor Κb ◽  
Mitogen Activated Protein Kinase ◽  
Host Recognition ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Opportunistic Human Pathogen

Fungi can shield surface pathogen-associated molecular patterns (PAMPs) for evading host immune attack. The most common and opportunistic human pathogen,Candida albicans, can shield β-(1 3)-glucan on the cell wall, one of the major PAMPs, to avoid host phagocyte Dectin-1 recognition. The way to interfere in the shielding process for more effective antifungal defense is not well established. In this study, we found that deletion of theC. albicansGPI7gene, which was responsible for adding ethanolaminephosphate to the second mannose in glycosylphosphatidylinositol (GPI) biosynthesis, could block the attachment of most GPI-anchored cell wall proteins (GPI-CWPs) to the cell wall and subsequently unmask the concealed β-(1,3)-glucan. Neutrophils could kill the uncloakedgpi7mutant more efficiently with an augmented respiratory burst. Thegpi7mutant also stimulated Dectin-1-dependent immune responses of macrophages, including activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways and secretion of specific cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-12p40. Furthermore, thegpi7null mutant could induce an enhanced inflammatory response through promoting significant recruitment of neutrophils and monocytes and could stimulate stronger Th1 and Th17 cell responses to fungal infectionsin vivo. Thesein vivophenotypes also were Dectin-1 dependent. Thus, we assume that GPI-CWPs are involved in the immune mechanism ofC. albicansescaping from host recognition by Dectin-1. Our studies also indicate that the blockage of GPI anchor synthesis is a strategy to inhibitC. albicansevading host recognition.

scholarly journals EspH Suppresses Erk by Spatial Segregation from CD81 Tetraspanin Microdomains

2018 ◽  
Vol 86 (10) ◽  
Author(s):  
Rachana Pattani Ramachandran ◽  
Felipe Vences-Catalán ◽  
Dan Wiseman ◽  
Efrat Zlotkin-Rivkin ◽  
Eyal Shteyer ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Inhibitory Effect ◽  
Effector Proteins ◽  
Mitogen Activated Protein Kinase ◽  
Erk Signaling ◽  
Human Pathogens ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
C Terminus ◽  
Tnf Α

ABSTRACT Enteropathogenic Escherichia coli (EPEC) belongs to a group of enteric human pathogens known as attaching-and-effacing (A/E) pathogens, which utilize a type III secretion system (T3SS) to translocate a battery of effector proteins from their own cytoplasm into host intestinal epithelial cells. Here we identified EspH to be an effector that prompts the recruitment of the tetraspanin CD81 to infection sites. EspH was also shown to be an effector that suppresses the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) signaling pathway at longer infection times. The inhibitory effect was abrogated upon deletion of the last 38 amino acids located at the C terminus of the protein. The efficacy of EspH-dependent Erk suppression was higher in CD81-deficient cells, suggesting that CD81 may act as a positive regulator of Erk, counteracting Erk suppression by EspH. EspH was found within CD81 microdomains soon after infection but was largely excluded from these domains at a later time. Based on our results, we propose a mechanism whereby CD81 is initially recruited to infection sites in response to EspH translocation. At a later stage, EspH moves out of the CD81 clusters to facilitate effective Erk inhibition. Moreover, EspH selectively inhibits the tumor necrosis factor alpha (TNF-α)-induced Erk signaling pathway. Since Erk and TNF-α have been implicated in innate immunity and cell survival, our studies suggest a novel mechanism by which EPEC suppresses these processes to promote its own colonization and survival in the infected gut.

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