Mutant forms of growth factor-binding protein-2 reverse BCR-ABL-induced transformation.

Growth factor-binding protein 2 (Grb2) is an adaptor protein that links tyrosine kinases to Ras. BCR-ABL is a tyrosine kinase oncoprotein that is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive leukemias. Grb2 forms a complex with BCR-ABL and the nucleotide exchange factor Sos that leads to the activation of the Ras protooncogene. In this report we demonstrate that Grb2 mutant proteins lacking amino- or carboxyl-terminal src homology SH3 domains suppress BCR-ABL-induced Ras activation and reverse the oncogenic phenotype. The Grb2 SH3-deletion mutant proteins bind to BCR-ABL and do not impair tyrosine kinase activity. Expression of the Grb2 SH3-deletion mutant proteins in BCR-ABL-transformed Rat-1 fibroblasts and in the human Ph1-positive leukemic cell line K562 inhibits their ability to grow as foci in soft agar and form tumors in nude mice. Furthermore, expression of the Grb2 SH3-deletion mutants in K562 cells induced their differentiation. Because Ras plays an important role in signaling by receptor and nonreceptor tyrosine kinases, the use of interfering mutant Grb2 proteins may be applied to block the proliferation of other cancers that depend in part on activated tyrosine kinases for growth.

Chemotactic signal integration in bacteria.

Chemotactic signaling in Escherichia coli involves transmission of both negative and positive signals. In order to examine mechanisms of signal processing, behavioral responses to dual inputs have been measured by using photoactivable "caged" compounds, computer video analysis, and chemoreceptor deletion mutants. Signaling from Tar and Tsr, two receptors that sense amino acids and pH, was studied. In a Tar deletion mutant the photoactivated release of protons, a Tsr repellent, and of serine, a Tsr attractant, in separate experiments at pH 7.0 resulted in tumbling (negative) or smooth-swimming (positive) responses in ca. 50 and 140 ms, respectively. Simultaneous photorelease of protons and serine resulted in a single tumbling or smooth-swimming response, depending on the relative amounts of the two effectors. In contrast, in wild-type E. coli, proton release at pH 7.0 resulted in a biphasic response that was attributed to Tsr-mediated tumbling followed by Tar-mediated smooth-swimming. In wild-type E. coli at more alkaline pH values the Tar-mediated signal was stronger than the Tsr signal, resulting in a strong smooth-swimming response preceded by a diminished tumbling response. These observations imply that (i) a single receptor time-averages the binding of different chemotactic ligands generating a single response; (ii) ligand binding to different receptors can result in a nonintegrated response with the tumbling response preceding the smooth-swimming response; (iii) however, chemotactic signals of different intensities derived from different receptors can also result in an apparently integrated response; and (iv) the different chemotactic responses to protons at neutral and alkaline pH may contribute to E. coli migration toward neutrality.

Identification of a viability domain in the granulocyte/macrophage colony-stimulating factor receptor beta-chain involving tyrosine-750.

The granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. In this study we have investigated domains of the GMR beta-chain (GMR beta) involved in maintaining cellular viability. Using a series of nested GMR beta deletion mutants, we demonstrate that there are at least two domains of GMR beta that contribute to viability signals. Deletion of amino acid residues 626-763 causes a viability defect that can be rescued with fetal calf serum (FCS). Deletion of residues 518-626, in contrast, causes a further decrement in viability that can be only partially compensated by the addition of FCS. GMR beta truncated proximal to amino acid 517 will not support long-term growth under any conditions. Site-directed mutagenesis of tyrosine-750 (Y750), which is contained within the distal viability domain, to phenylalanine eliminates all demonstrable tyrosine phosphorylation of GMR beta. Cell lines transfected with mutant GMR beta (Y750-->F) have a viability disadvantage when compared to cell lines containing wild-type GMR that is partially rescued by the addition of FCS. We studied signal transduction in mutant cell lines in an effort to identify pathways that might participate in the viability signal. Although tyrosine phosphorylation of JAK2, SHPTP2, and Vav is intact in Y750-->F mutant cell lines, Shc tyrosine phosphorylation is reduced. This suggests a potential role for Y750 and potentially Shc in a GM-CSF-induced signaling pathway that helps maintain cellular viability.

Glycosylation of the c-Myc transactivation domain.

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic posttranslational modification composed of a single monosaccharide, GlcNAc, glycosidically composed of a single monosaccharide, GlcNAc, glycosidically linked to the side-chain hydroxyl of serine or threonine residues. Although O-GlcNAc occurs on a myriad of nuclear and cytoplasmic proteins, only a few have thus far been identified. These O-GlcNAc-bearing proteins are also modified by phosphorylation and form reversible multimeric complexes. Here we present evidence for O-GlcNAc glycosylation of the oncoprotein c-Myc, a helix-loop-helix/leucine zipper phosphoprotein that heterodimerizes with Max and participates in the regulation of gene transcription in normal and neoplastic cells. O-GlcNAc modification of c-Myc is shown by three different methods: (i) demonstration of lectin binding to in vitro translated protein using a protein-protein interaction mobility-shift assay; (ii) glycosidase or glycosyltransferase treatment of in vitro translated protein analyzed by lectin affinity chromatography; and (iii) direct characterization of the sugar moieties on purified recombinant protein overexpressed in either insect cells or Chinese hamster ovary cells. Analyses of serial deletion mutants of c-Myc further suggest that the O-GlcNAc site(s) are located within or near the N-terminal transcription activation/malignant transformation domain, a region where mutations of c-Myc that are frequently found in Burkitt and AIDS-related lymphomas cluster.

The Epstein-Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein

The Epstein-Barr virus nuclear antigen (EBNA)-6 protein is essential for Epstein-Barr virus (EBV)-induced immortalization of primary human B-lymphocytes in vitro. In this study, fusion proteins of EBNA-6 with green fluorescent protein (GFP) have been used to characterize its nuclear localization and organization within the nucleus. EBNA-6 associates with nuclear structures and in immunofluorescence demonstrate a punctate staining pattern. Herein, we show that the association of EBNA-6 with these nuclear structures was maintained throughout the cell cycle and with the use of GFP-E6 deletion mutants, that the region amino acids 733-808 of EBNA-6 contains a domain that can influence the association of EBNA-6 with these nuclear structures. Co-immunofluorescence and confocal analyses demonstrated that EBNA-6 and EBNA-3 co-localize in the nucleus of cells. Expression of EBNA-6, but not EBNA-3, caused a redistribution of nuclear survival of motor neurons protein (SMN) to the EBNA-6 containing nuclear structures resulting in co-localization of SMN with EBNA-6. (C) 2003 Elsevier Inc. All rights reserved.

Molecular analysis of the psa permease complex of Streptococcus pneumoniae

The psaBCA locus of Streptococcus pneumoniae encodes a putative ABC Mn2+-permease complex. Downstream of the operon is psaD, which may be co-transcribed and encodes a thiol peroxidase. Previously, there has been discordance concerning the phenotypic impact of mutations in the psa locus, resolution of which has been complicated by differences in mutant construction and the possibility of polar effects. Here, we constructed unmarked, in frame deletion mutants DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaD, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD in S. pneumoniae D39 to examine the role of each gene within the locus in Mn2+ uptake, susceptibility to oxidative stress, virulence, nasopharyngeal colonization and chain morphology. The requirement for Mn2+ for growth and transformation was also investigated for all mutants. Inductively coupled plasma mass spectrometry (ICP-MS) analysis provided the first direct evidence that PsaBCA is indeed a Mn2+ transporter. However, this study did not substantiate previous reports that the locus plays a role in choline-binding protein pro-duction or chain morphology. We also confirmed the importance of the Psa permease in systemic virulence and resistance to superoxide and hydrogen peroxide, as well as demonstrating a role in nasopharyngeal colonization for the first time. Further evi-dence is provided to support the requirement for Mn2+ supplementation for growth and transformation of DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD mutants. However, transformation, as well as growth, of the DeltapsaD mutant was not dependent upon Mn2+ supplementation. We also show that, apart from sensitivity to hydrogen peroxide, the DeltapsaD mutant exhibited essentially similar phenotypes to those of the wild type. Western blot analysis with a PsaD antiserum showed that deleting any of the genes upstream of psaD did not affect its expression. However, we found that deleting psaB resulted in decreased expression of PsaA relative to that in D39, whereas deleting both psaB and psaC resulted in at least wild-type levels of PsaA.

Association of stomatin with lipid bodies

The oligomeric lipid raft-associated integral protein stomatin normally localizes to the plasma membrane and the late endosomal compartment. Similar to the caveolins, it is targeted to lipid bodies (LBs) on overexpression. Endogenous stomatin also associates with LBs to a small extent. Green fluorescent protein-tagged stomatin (StomGFP) and the dominant-negative caveolin-3 mutant DGV(cav3)(HA) occupy distinct domains on LB surfaces but eventually intermix. Studies of StomGFP deletion mutants reveal that the region for membrane association but not oligomerization and raft association is essential for LB targeting. Blocking protein synthesis leads to the redistribution of StomGFP from LBs to LysoTracker-positive vesicles indicating a connection with the late endosomal/ lysosomal pathway. Live microscopy of StomGFP reveals multiple interactions between LBs and microtubule-associated vesicles possibly representing signaling events and/or the exchange of cargo. Proteomic analysis of isolated LBs identifies adipophilin and TIP47, various lipid-specific enzymes, cytoskeletal components, chaperones, Ras-related proteins, protein kinase D2, and other regulatory proteins. The association of the Rab proteins 1, 6, 7, 10, and 18 with LBs indicates various connections to other compartments. Our data suggest that LBs are not only involved in the storage of lipids but also participate actively in the cellular signaling network and the homeostasis of lipids.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H+-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

Factors important to the secretion and matrix deposition of tissue transglutaminase

Data suggest that for TG2 to be secreted, an intact N-terminal FN binding site (for which TG2 has high affinity) is required, however interaction of TG2 with its high affinity binding partners presents both in the intracellular and extracellular space as well as with specific cell surface receptors may also be involved in this process. Using a site-directed mutagenesis approach, the effects of specific mutations of TG2 on its translocation to the cell surface and secretion into the ECM have been investigated. Mutations include those affecting FN binding (FN1), HSPGs binding (HS1, HS2) GTP/GDP binding site (GTP1, 2) as well as N-terminal and C-terminal domains (TG2 deletion mutants N, and C). By performing transglutaminase activity assays, cell surface protein biotinylation and verifying distribution of TG2 mutants in the ECM we demonstrated that one of the potential heparan sulfate binding site mutants (HS2 mutant) is secreted at the cell surface in a much reduced manner and is less deposited into the ECM than the HS1 mutant. The HS2 mutant showed a low affinity for binding to a heparin sepharose column demonstrating this mutation site may be a potential heparan binding site of TG2. Analogous peptides to this site were shown to have some efficiency in the inhibition of the binding of the FN-TG2 complex to cell surface heparan sulfates in a cell adhesion assay indicating the peptide to be representative of the novel heparin binding site within TG2. The GTP binding site mutants GTP1 and GTP2 exhibited low specific activity however, GTP2 showed more secretion to the cell surface in comparison to GTP1. The FN1 binding mutant did not greatly affect TG2 activity nor did it alter TG2 secretion at the cell surface and deposition into the ECM indicating that fibronectin binding at this site on the enzyme is not an important factor. Interestingly an intact N-terminus (?1-15) appeared to be essential for enzyme externalisation. Removal of the first 15 amino acids (N-terminal mutant) abolished TG2 secretion to the cell surface as well as deposition into the ECM. In addition it reduced the enzymes affinity for binding to heparin. In contrast, deletion of the C-terminal TG2 domain (?594-687) increased enzyme secretion to the cell surface. Consistent with the data presented in this thesis we speculate that TG2 must fulfill two requirements to be successfully secreted from cells. The findings indicate that the closed conformation of the enzyme as well as intact N-terminal tail and a novel HS binding site within the TG2 molecule are key elements for the enzyme’s localisation at the cell surface and its deposition into the extracellular matrix. The importance of understanding the interactions between TG2, heparan sulfates and other TG2 binding partners at the cell surface could have an impact on the design of novel strategies for enzyme inhibition which could be important in the control of extracellular TG2 related diseases.

Role of Pseudomonas aeruginosa mifSR Two-Component System in Antibiotic Resistance and Biofilm Formation

Pseudomonas aeruginosa is an opportunistic pathogen found in a wide variety of environments. It is one of the leading causes of morbidity and mortality in cystic fibrosis patients, and one of the main sources of nosocomial infections in the United States. One of the most prominent features of this pathogen is its wide resistance to antibiotics. P. aeruginosa employs a variety of mechanisms including efflux pumps and the expression of B-lactamases to overcome antibiotic treatment. Two chromosomally encoded lactamases, ampC and poxB, have been identified in P. aeruginosa. Sequence analyses have shown the presence of a two-component system (TCS) called MifSR (MifS-Sensor and MifR-Response Regulator), immediately upstream of the poxAB operon. It is hypothesized that the MifSR TCS is involved in B-lactam resistance via the regulation of poxB. Recently, the response regulator MifR has been reported to play a crucial role in biofilm formation, a major characteristic of chronic infections and increased antibiotic resistance. In this study, mifR and mifSR deletion mutants were constructed, and compared to the wild type parent strain PAOl for differences in growth and B-lactam sensitivity. Results obtained thus far indicate that mifR and mifSR are not essential for growth, and do not confer B-lactam resistance under the conditions tested. This study is significant because biofilm formation and antibiotic resistance are two hallmarks of P. aeruginosa infections, and finding a link between these two may lead to the development of improved treatment strategies.

Spontaneous Unfolding and Refolding of FNIII Domains Assayed by Thiol Exchange

Fibronectin (FN) is a large extracellular matrix (ECM) protein that is made up of

type I (FNI), type II (FNII), & type III (FNIII) domains. It assembles into an insoluble

supra-­‐‑molecular structure: the fibrillar FN matrix. FN fibrillogenesis is a cell‐‑mediated process, which is initiated when FN binds to integrins on the cell surface. The FN matrix plays an important role in cell migration, proliferation, signaling & adhesion. Despite decades of research, the FN matrix is one of the least understood supra-­‐‑molecular protein assemblies. There have been several attempts to elucidate the exact mechanism of matrix assembly resulting in significant progress in the field but it is still unclear as to what are FN-­‐‑FN interactions, the nature of these interactions and the domains of FN that

are in contact with each other. FN matrix fibrils are elastic in nature. Two models have been proposed to explain the elasticity of the fibrils. The first model: the ‘domain unfolding’ model postulates that the unraveling of FNIII domains under tension explains fibril elasticity.

The second model relies on the conformational change of FN from compact to extended to explain fibril elasticity. FN contain 15 FNIII domains, each a 7-­‐‑strand beta sandwich. Earlier work from our lab used the technique of labeling a buried Cys to study the ‘domain unfolding’ model. They used mutant FNs containing a buried Cys in a single FNIII domain and found that 6 of the 15 FNIII domains label in matrix fibrils. Domain unfolding due to tension, matrix associated conformational changes or spontaneous folding and unfolding are all possible explanation for labeling of the buried Cys. The present study also uses the technique of labeling a buried Cys to address whether it is spontaneous folding and unfolding that labels FNIII domains in cell culture. We used thiol reactive DTNB to measure the kinetics of labeling of buried Cys in eleven FN III domains over a wide range of urea concentrations (0-­‐‑9M). The kinetics data were globally fit using Mathematica. The results are equivalent to those of H-­‐‑D exchange, and

provide a comprehensive analysis of stability and unfolding/folding kinetics of each

domain. For two of the six domains spontaneous folding and unfolding is possibly the reason for labeling in cell culture. For the rest of the four domains it is probably matrix associated conformational changes or tension induced unfolding.

A long-­‐‑standing debate in the protein-­‐‑folding field is whether unfolding rate

constants or folding rate constants correlate to the stability of a protein. FNIII domains all have the same ß sandwich structure but very different stabilities and amino acid sequences. Our study analyzed the kinetics of unfolding and folding and stabilities of eleven FNIII domains and our results show that folding rate constants for FNIII domains are relatively similar and the unfolding rates vary widely and correlate to stability. FN forms a fibrillar matrix and the FN-­‐‑FN interactions during matrix fibril formation are not known. FNI 1-­‐‑9 or the N-­‐‑ terminal region is indispensible for matrix formation and its major binding partner has been shown to be FNIII 2. Earlier work from our lab, using FRET analysis showed that the interaction of FNI 1-­‐‑9 with a destabilized FNIII 2 (missing the G strand, FNIII 2ΔG) reduces the FRET efficiency. This efficiency is restored in the presence of FUD (bacterial adhesion from S. pyogenes) that has been known to interact with FNI 1-­‐‑9 via a tandem ß zipper. In the present study we

use FRET analysis and a series of deletion mutants of FNIII 2ΔG to study the shortest fragment of FNIII 2ΔG that is required to bind FNI 1-­‐‑9. Our results presented here are qualitative and show that FNIII 2ΔC’EFG is the shortest fragment required to bind FNI 1-­‐‑9. Deletion of one more strand abolishes the interaction with FNI 1-­‐‑9.

Characterization of Borrelia burgdorferi Gene Product BBD18 for Its Role(s) in Pathogen Biology and Infectivity

bbd18 is a differentially expressed Borrelia burgdorferi gene that is transcribed at almost undetectable levels in spirochetes grown in vitro but dramatically upregulated during tick infection. The gene also displays low yet detectable expression at various times in tissues of murine hosts. As the gene product bears no homology to known proteins, its biological significance remains enigmatic. To understand the gene function, we created isogenic bbd18-deletion mutants as well as genetically-complemented isolates from an infectious wild-type B. burgdorferi strain. Compared to parental isolates, bbd18 mutants - but not complemented spirochetes - displayed slower in vitro growth. The bbd18 mutants also reflect significantly reduced ability to persist or remain undetectable both in immunocompetent and SCID mice, yet were able to survive in ticks. This suggests BBD18 function is essential in mammalian hosts but redundant in the arthropod vector. Notably, although bbd18 expression and in vitro growth defects are restored in the complemented isolates, their phenotype is similar to the mutants - being unable to persist in mice but able to survive in ticks. Despite low expression in cultured wild-type B. burgdorferi, bbd18 deletion downregulated several genes. Interestingly, expression of some, including ospD and bbi39, could be complemented, while that of others could not be restored via bbd18 re-expression. Correspondingly, bbd18 mutants displayed altered production of several proteins, and similar to RNA levels, some were restored in the bbd18 complement and others not. To understand how bbd18 deletion results in apparently permanent and noncomplementable phenotypic defects, we sought to genetically disturb the DNA topology surrounding the bbd18 locus without deleting the gene. Spirochetes with an antibiotic cassette inserted downstream of the gene, between bbd17 and bbd18, were significantly attenuated in mice, while a similar upstream insertion, between bbd18 and bbd19, did not affect infectivity, suggesting that an unidentified cis element downstream of bbd18 may encode a virulence-associated factor critical for infection.

Molecular chaperone accumulation as a function of stress evidences adaptation to high hydrostatic pressure in the piezophilic archaeon Thermococcus barophilus

The accumulation of mannosyl-glycerate (MG), the salinity stress response osmolyte of Thermococcales, was investigated as a function of hydrostatic pressure in Thermococcus barophilus strain MP, a hyperthermophilic, piezophilic archaeon isolated from the Snake Pit site (MAR), which grows optimally at 40 MPa. Strain MP accumulated MG primarily in response to salinity stress, but in contrast to other Thermococcales, MG was also accumulated in response to thermal stress. MG accumulation peaked for combined stresses. The accumulation of MG was drastically increased under sub-optimal hydrostatic pressure conditions, demonstrating that low pressure is perceived as a stress in this piezophile, and that the proteome of T. barophilus is low-pressure sensitive. MG accumulation was strongly reduced under supra-optimal pressure conditions clearly demonstrating the structural adaptation of this proteome to high hydrostatic pressure. The lack of MG synthesis only slightly altered the growth characteristics of two different MG synthesis deletion mutants. No shift to other osmolytes was observed. Altogether our observations suggest that the salinity stress response in T. barophilus is not essential and may be under negative selective pressure, similarly to what has been observed for its thermal stress response. Introduction

Functional and structural dissection of the tape measure protein of lactococcal phage TP901-1

The tail tape measure protein (TMP) of tailed bacteriophages (also called phages) dictates the tail length and facilitates DNA transit to the cell cytoplasm during infection. Here, a thorough mutational analysis of the TMP from lactococcal phage TP901-1 (TMPTP901-1) was undertaken. We generated 56 mutants aimed at defining TMPTP901-1 domains that are essential for tail assembly and successful infection. Through analysis of the derived mutants, we determined that TP901-1 infectivity requires the N-terminal 154 aa residues, the C-terminal 60 residues and the first predicted hydrophobic region of TMPTP901-1 as a minimum. Furthermore, the role of TMPTP901-1 in tail length determination was visualized by electron microscopic imaging of TMP-deletion mutants. The inverse linear correlation between the extent of TMPTP901-1-encoding gene deletions and tail length of the corresponding virion provides an estimate of TMPTP901-1 regions interacting with the connector or involved in initiator complex formation. This study represents the most thorough characterisation of a TMP from a Gram-positive host-infecting phage and provides essential advances to understanding its role in virion assembly, morphology and infection.

Investigating the mechanisms of Moraxella catarrhalis resistance to oxidative stress

Moraxella catarrhalis (Mcat) represents a human pathogen implicated in debilitating diseases, such as Chronic Obstructive Pulmonary Disease (COPD). One of the hallmarks of COPD is the excessive neutrophil oxidative stress mediated by reactive oxygen species (ROS). Mcat shows a higher innate level of resistance to exogenous oxidative stress compared to the co-infecting human airways pathogens such as non-typeable Haemophilus influenzae (NTHi) but the underlying mechanisms are currently not well defined. In this thesis, we demonstrated that, differently from NTHi, Mcat was able to directly interfere with ROS production and ROS-related responses such as neutrophil extracellular traps (NET) and autophagy in differentiated neutrophilic-like dHL-60 cells and primary cells. The underlying mechanisms were shown to be phagocytosis/opsonins-independent but contact-dependent, due to the engagement of the immunosuppressive receptors. Indeed, we identified that through OmpCD porin, Mcat was able to engage Siglec inhibitory receptors suppressing ROS generation by the host cells. Furthermore, Mcat provided a safer niche for the co-infecting NTHi bacterium which was otherwise susceptible to the host antimicrobial arsenal. Subsequently, to deeply characterize the Mcat global transcriptional response to oxidative stress, an RNA-Seq experiment was performed on exponentially growing bacteria exposed to sublethal amounts of H2O2 or CuSO4, stimuli that the pathogens experienced once they are phagocytosed. We unraveled a previously unidentified common transcriptional program following H2O2 and CuSO4 exposure, demonstrating a similar defense mechanism to the stress conditions encountered in neutrophils. We ascertained new crucial factors for this pathogen response and established a novel in vivo Mcat infection model, using the invertebrate Galleria mellonella. Actually, we observed that deletion mutants of genes implicated in oxidative stress resistance exhibited reduced virulence. In conclusion, this work represents an important step in the understanding of Mcat innate resistance mechanisms to oxidative stress and further elucidate the virulence mechanisms during infection.