scholarly journals Vertical Transmission at the Pathogen-Symbiont Interface: Serratia symbiotica and Aphids

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Julie Perreau ◽  
Devki J. Patel ◽  
Hanna Anderson ◽  
Gerald P. Maeda ◽  
Katherine M. Elston ◽  
...  
Keyword(s):  
Vertical Transmission ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Experimental Studies ◽  
Early Embryo ◽  
Host Cells ◽  
Bacterial Symbionts ◽  
Content Type ◽  
Serratia Symbiotica ◽  
Gut Pathogens

ABSTRACT Many insects possess beneficial bacterial symbionts that occupy specialized host cells and are maternally transmitted. As a consequence of their host-restricted lifestyle, these symbionts often possess reduced genomes and cannot be cultured outside hosts, limiting their study. The bacterial species Serratia symbiotica was originally characterized as noncultured strains that live as mutualistic symbionts of aphids and are vertically transmitted through transovarial endocytosis within the mother’s body. More recently, culturable strains of S. symbiotica were discovered that retain a larger set of ancestral Serratia genes, are gut pathogens in aphid hosts, and are principally transmitted via a fecal-oral route. We find that these culturable strains, when injected into pea aphids, replicate in the hemolymph and are pathogenic. Unexpectedly, they are also capable of maternal transmission via transovarial endocytosis: using green fluorescent protein (GFP)-tagged strains, we observe that pathogenic S. symbiotica strains, but not Escherichia coli, are endocytosed into early embryos. Furthermore, pathogenic S. symbiotica strains are compartmentalized into specialized aphid cells in a fashion similar to that of mutualistic S. symbiotica strains during later stages of embryonic development. However, infected embryos do not appear to develop properly, and offspring infected by a transovarial route are not observed. Thus, cultured pathogenic strains of S. symbiotica have the latent capacity to transition to lifestyles as mutualistic symbionts of aphid hosts, but persistent vertical transmission is blocked by their pathogenicity. To transition into stably inherited symbionts, culturable S. symbiotica strains may need to adapt to regulate their titer, limit their pathogenicity, and/or provide benefits to aphids that outweigh their cost. IMPORTANCE Insects have evolved various mechanisms to reliably transmit their beneficial bacterial symbionts to the next generation. Sap-sucking insects, including aphids, transmit symbionts by endocytosis of the symbiont into cells of the early embryo within the mother’s body. Experimental studies of this process are hampered by the inability to culture or genetically manipulate host-restricted, symbiotic bacteria. Serratia symbiotica is a bacterial species that includes strains ranging from obligate, heritable symbionts to gut pathogens. We demonstrate that culturable S. symbiotica strains, which are aphid gut pathogens, can be maternally transmitted. Cultured S. symbiotica therefore possesses a latent capacity for evolving a host-restricted lifestyle and can be used to understand the transition from pathogenicity to beneficial symbiosis.

scholarly journals Vertical transmission at the pathogen-symbiont interface: Serratia symbiotica and aphids

2020 ◽  
Author(s):  
Julie Perreau ◽  
Devki J. Patel ◽  
Hanna Anderson ◽  
Gerald P. Maeda ◽  
Katherine M. Elston ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Experimental Studies ◽  
Oral Route ◽  
Early Embryo ◽  
Host Cells ◽  
Bacterial Symbionts ◽  
Early Embryos ◽  
Pea Aphids ◽  
Serratia Symbiotica ◽  
Gut Pathogens

AbstractMany insects possess beneficial bacterial symbionts that occupy specialized host cells and are maternally transmitted. As a consequence of their host-restricted lifestyle, these symbionts often possess reduced genomes and cannot be cultured outside hosts, limiting their study. The bacterial species Serratia symbiotica was originally described by noncultured strains that live as mutualistic symbionts of aphids and are vertically transmitted through transovarial endocytosis within the mother’s body. More recently, culturable strains of S. symbiotica were discovered that retain a larger set of ancestral Serratia genes, are gut pathogens in aphid hosts, and are principally transmitted via a fecal-oral route. We find that these culturable strains, when injected into pea aphids, replicate in the hemolymph and are pathogenic. Unexpectedly, they are also capable of maternal transmission via transovarial endocytosis: using GFP-tagged strains, we observe that pathogenic S. symbiotica, but not Escherichia coli, are endocytosed into early embryos. Furthermore, pathogenic S. symbiotica strains are compartmentalized into specialized aphid cells in a similar fashion to mutualistic S. symbiotica strains during later stages of embryonic development. Thus, cultured, pathogenic strains of S. symbiotica have the latent capacity to transition to lifestyles as mutualistic symbionts of aphid hosts. This capacity is blocked by pathogenicity: their hosts die before infected progeny are born. To transition into stably inherited symbionts, culturable S. symbiotica strains may need to adapt to regulate their titer, limit their pathogenicity, and/or provide benefits to aphids that outweigh their cost.ImportanceInsects have evolved various mechanisms to reliably transmit their beneficial bacterial symbionts to the next generation. Sap-sucking insects, including aphids, transmit symbionts by endocytosis of the symbiont into cells of the early embryo within the mother’s body. Experimental studies of this process are hampered by the inability to culture or genetically manipulate host-restricted, symbiotic bacteria. Serratia symbiotica is a bacterial species that includes strains ranging from obligate, heritable symbionts to culturable gut pathogens. We demonstrate that culturable S. symbiotica strains, that are aphid gut pathogens, can be maternally transmitted by endocytosis. Cultured S. symbiotica therefore possess a latent capacity for evolving a host-restricted lifestyle and can be used to understand the transition from pathogenicity to beneficial symbiosis.

scholarly journals hfqPlays Important Roles in Virulence and Stress Adaptation in Cronobacter sakazakii ATCC 29544

2015 ◽  
Vol 83 (5) ◽  
pp. 2089-2098 ◽  
Author(s):  
Seongok Kim ◽  
Hyelyeon Hwang ◽  
Kwang-Pyo Kim ◽  
Hyunjin Yoon ◽  
Dong-Hyun Kang ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Soft Agar ◽  
Host Cells ◽  
Neonatal Meningitis ◽  
Animal Cells ◽  
Content Type ◽  
Flagellar Biosynthesis

Cronobacterspp. are opportunistic pathogens that cause neonatal meningitis and sepsis with high mortality in neonates. Despite the peril associated withCronobacterinfection, the mechanisms of pathogenesis are still being unraveled. Hfq, which is known as an RNA chaperone, participates in the interaction with bacterial small RNAs (sRNAs) to regulate posttranscriptionally the expression of various genes. Recent studies have demonstrated that Hfq contributes to the pathogenesis of numerous species of bacteria, and its roles are varied between bacterial species. Here, we tried to elucidate the role of Hfq inC. sakazakiivirulence. In the absence ofhfq,C. sakazakiiwas highly attenuated in disseminationin vivo, showed defects in invasion (3-fold) into animal cells and survival (103-fold) within host cells, and exhibited low resistance to hydrogen peroxide (102-fold). Remarkably, the loss ofhfqled to hypermotility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyperflagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagellar biosynthesis in a strain lackinghfq. Together, these data strongly suggest thathfqplays important roles in the virulence ofC. sakazakiiby participating in the regulation of multiple genes.

scholarly journals Novel Model for Multispecies Biofilms That Uses Rigid Gas-Permeable Lenses

2011 ◽  
Vol 77 (10) ◽  
pp. 3413-3421 ◽  
Author(s):  
Rebecca Peyyala ◽  
Sreenatha S. Kirakodu ◽  
Jeffrey L. Ebersole ◽  
Karen F. Novak
Keyword(s):  
Laser Scanning ◽  
Bacterial Species ◽  
Disease Process ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Laser ◽  
Content Type ◽  
Oral Biofilms ◽  
Multispecies Biofilms

ABSTRACTOral biofilms comprise complex multispecies consortia aided by specific inter- and intraspecies interactions occurring among commensals and pathogenic bacterial species. Oral biofilms are primary initiating factors of periodontal disease, although complex multifactorial biological influences, including host cell responses, contribute to the individual outcome of the disease. To provide a system to study initial stages of interaction between oral biofilms and the host cells that contribute to the disease process, we developed a novelin vitromodel system to grow biofilms on rigid gas-permeable contact lenses (RGPLs), which enable oxygen to permeate through the lens material. Bacterial species belonging to early- and late-colonizing groups were successfully established as single- or three-species biofilms, with each group comprisingStreptococcus gordonii,Streptococcus oralis, andStreptococcus sanguinis;S. gordonii,Actinomyces naeslundii, andFusobacterium nucleatum; orS. gordonii,F. nucleatum, andPorphyromonas gingivalis. Quantification of biofilm numbers by quantitative PCR (qPCR) revealed substantial differences in the magnitude of bacterial numbers in single-species and multispecies biofilms. We evaluated cell-permeable conventional nucleic acid stains acridine orange, hexidium iodide, and Hoechst 33258 and novel SYTO red, blue, and green fluorochromes for their effect on bacterial viability and fluorescence yield to allow visualization of the aggregates of individual bacterial species by confocal laser scanning microscopy (CLSM). Substantial differences in the quantity and distribution of the species in the multispecies biofilms were identified. The specific features of these biofilms may help us better understand the role of various bacteria in local challenge of oral tissues.

scholarly journals Two Bacterial Genera, Sodalis and Rickettsia, Associated with the Seal Louse Proechinophthirus fluctus (Phthiraptera: Anoplura)

2016 ◽  
Vol 82 (11) ◽  
pp. 3185-3197 ◽  
Author(s):  
Bret M. Boyd ◽  
Julie M. Allen ◽  
Ryuichi Koga ◽  
Takema Fukatsu ◽  
Andrew D. Sweet ◽  
...  
Keyword(s):  
Vertical Transmission ◽  
Marine Mammals ◽  
Bacterial Symbiont ◽  
Symbiotic Bacteria ◽  
Bacterial Symbionts ◽  
Human Pathogen ◽  
Content Type ◽  
Fur Seals ◽  
Symbiont Genome ◽  
Blood Meals

ABSTRACTRoughly 10% to 15% of insect species host heritable symbiotic bacteria known as endosymbionts. The lice parasitizing mammals rely on endosymbionts to provide essential vitamins absent in their blood meals. Here, we describe two bacterial associates from a louse,Proechinophthirus fluctus, which is an obligate ectoparasite of a marine mammal. One of these is a heritable endosymbiont that is not closely related to endosymbionts of other mammalian lice. Rather, it is more closely related to endosymbionts of the genusSodalisassociated with spittlebugs and feather-chewing bird lice. Localization and vertical transmission of this endosymbiont are also more similar to those of bird lice than to those of other mammalian lice. The endosymbiont genome appears to be degrading in symbiosis; however, it is considerably larger than the genomes of other mammalian louse endosymbionts. These patterns suggest the possibility that thisSodalisendosymbiont might be recently acquired, replacing a now-extinct, ancient endosymbiont. From the same lice, we also identified an abundant bacterium belonging to the genusRickettsiathat is closely related toRickettsia ricketsii, a human pathogen vectored by ticks. No obvious masses of theRickettsiabacterium were observed in louse tissues, nor did we find any evidence of vertical transmission, so the nature of its association remains unclear.IMPORTANCEMany insects are host to heritable symbiotic bacteria. These heritable bacteria have been identified from numerous species of parasitic lice. It appears that novel symbioses have formed between lice and bacteria many times, with new bacterial symbionts potentially replacing existing ones. However, little was known about the symbionts of lice parasitizing marine mammals. Here, we identified a heritable bacterial symbiont in lice parasitizing northern fur seals. This bacterial symbiont appears to have been recently acquired by the lice. The findings reported here provide insights into how new symbioses form and how this lifestyle is shaping the symbiont genome.

scholarly journals Yersinia pestis YopK Inhibits Bacterial Adhesion to Host Cells by Binding to the Extracellular Matrix Adaptor Protein Matrilin-2

2017 ◽  
Vol 85 (8) ◽  
Author(s):  
Yafang Tan ◽  
Wanbing Liu ◽  
Qingwen Zhang ◽  
Shiyang Cao ◽  
Haihong Zhao ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hela Cells ◽  
Bacterial Adhesion ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Adaptor Protein ◽  
Innate Immune ◽  
Host Cells ◽  
Content Type ◽  
Green Fluorescent

ABSTRACT Pathogenic yersiniae harbor a type III secretion system (T3SS) that injects Yersinia outer protein (Yop) into host cells. YopK has been shown to control Yop translocation and prevent inflammasome recognition of the T3SS by the innate immune system. Here, we demonstrate that YopK inhibits bacterial adherence to host cells by binding to the extracellular matrix adaptor protein matrilin-2 (MATN2). YopK binds to MATN2, and deleting amino acids 91 to 124 disrupts binding of YopK to MATN2. A yopK null mutant exhibits a hyperadhesive phenotype, which could be responsible for the established Yop hypertranslocation phenotype of yopK mutants. Expression of YopK, but not YopKΔ91–124, in a yopK mutant restored the wild-type phenotypes of adhesion and Yop translocation, suggesting that binding to MATN2 might be essential for YopK to inhibit bacterial adhesion and negatively regulate Yop translocation. A green fluorescent protein (GFP)-YopK fusion specifically binds to the endogenous MATN2 on the surface of HeLa cells, whereas GFP-YopKΔ91–124 cannot. Addition of purified YopK protein during infection decreased adhesion of Y. pestis to HeLa cells, while YopKΔ91–124 protein showed no effect. Taking these results together, we propose a model that the T3SS-secreted YopK hinders bacterial adhesion to HeLa cells by binding to MATN2, which is ubiquitously exposed on eukaryotic cells.

scholarly journals The Coat Protein and NIa Protease of Two Potyviridae Family Members Independently Confer Superinfection Exclusion

2016 ◽  
Vol 90 (23) ◽  
pp. 10886-10905 ◽  
Author(s):  
Satyanarayana Tatineni ◽  
Roy French
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Wheat Streak Mosaic Virus ◽  
Host Cells ◽  
Content Type ◽  
Transgenic Expression ◽  
Superinfection Exclusion ◽  
Functional Conservation ◽  
Virus Interaction

ABSTRACTSuperinfection exclusion (SIE) is an antagonistic virus-virus interaction whereby initial infection by one virus prevents subsequent infection by closely related viruses. Although SIE has been described in diverse viruses infecting plants, humans, and animals, its mechanisms, including involvement of specific viral determinants, are just beginning to be elucidated. In this study, SIE determinants encoded by two economically important wheat viruses,Wheat streak mosaic virus(WSMV; genusTritimovirus, familyPotyviridae) andTriticum mosaic virus(TriMV; genusPoacevirus, familyPotyviridae), were identified in gain-of-function experiments that used heterologous viruses to express individual virus-encoded proteins in wheat. Wheat plants infected with TriMV expressing WSMV P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, or NIb cistrons permitted efficient superinfection by WSMV expressing green fluorescent protein (WSMV-GFP). In contrast, wheat infected with TriMV expressing WSMV NIa-Pro or coat protein (CP) substantially excluded superinfection by WSMV-GFP, suggesting that both of these cistrons are SIE effectors encoded by WSMV. Importantly, SIE is due to functional WSMV NIa-Pro or CP rather than their encoding RNAs, as altering the coded protein products by minimally changing RNA sequences led to abolishment of SIE. Deletion mutagenesis further revealed that elicitation of SIE by NIa-Pro requires the entire protein while CP requires only a 200-amino-acid (aa) middle fragment (aa 101 to 300) of the 349 aa. Strikingly, reciprocal experiments with WSMV-mediated expression of TriMV proteins showed that TriMV CP, and TriMV NIa-Pro to a lesser extent, likewise excluded superinfection by TriMV-GFP. Collectively, these data demonstrate that WSMV- and TriMV-encoded CP and NIa-Pro proteins are effectors of SIE and that these two proteins trigger SIE independently of each other.IMPORTANCESuperinfection exclusion (SIE) is an antagonistic virus-virus interaction that prevents secondary invasions by identical or closely related viruses in the same host cells. Although known to occur in diverse viruses, SIE remains an enigma in terms of key molecular determinants and action mechanisms. In this study, we found thatWheat streak mosaic virus(WSMV) andTriticum mosaic virus(TriMV) encode two independently functioning cistrons that serve as effectors of SIE at the protein but not the RNA level. The coat protein and NIa-Pro encoded by these two viruses, when expressed from a heterologous virus, exerted SIE to the cognate viruses. The identification of virus-encoded effectors of SIE and their transgenic expression could potentially facilitate the development of virus-resistant crop plants. Additionally, functional conservation of SIE in diverse virus groups suggests that a better understanding of the underlying mechanisms of SIE could facilitate the development of novel antiviral therapies against viral diseases.

scholarly journals Type II Secretion Promotes Bacterial Growth within the Legionella-Containing Vacuole in Infected Amoebae

2019 ◽  
Vol 87 (11) ◽  
Author(s):  
Richard C. White ◽  
Hilary K. Truchan ◽  
Huaixin Zheng ◽  
Jessica Y. Tyson ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Recent Analysis ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Macrophage Infection ◽  
Content Type ◽  
Intracellular Infection

ABSTRACT It was previously determined that the type II secretion system (T2SS) promotes the ability of Legionella pneumophila to grow in coculture with amoebae. Here, we discerned the stage of intracellular infection that is potentiated by comparing the wild-type and T2SS mutant legionellae for their capacity to parasitize Acanthamoeba castellanii. Whereas the mutant behaved normally for entry into the host cells and subsequent evasion of degradative lysosomes, it was impaired in the ability to replicate, with that defect being first evident at approximately 9 h postentry. The replication defect was initially documented in three ways: by determining the numbers of CFU recovered from the lysates of the infected monolayers, by monitoring the levels of fluorescence associated with amoebal monolayers infected with green fluorescent protein (GFP)-expressing bacteria, and by utilizing flow cytometry to quantitate the amounts of GFP-expressing bacteria in individual amoebae. By employing confocal microscopy and newer imaging techniques, we further determined the progression in volume and shape of the bacterial vacuoles and found that the T2SS mutant grows at a decreased rate and does not attain maximally sized phagosomes. Overall, the entire infection cycle (i.e., entry to egress) was considerably slower for the T2SS mutant than it was for the wild-type strain, and the mutant’s defect was maintained over multiple rounds of infection. Thus, the T2SS is absolutely required for L. pneumophila to grow to larger numbers in its intravacuolar niche within amoebae. Combining these results with those of our recent analysis of macrophage infection, T2SS is clearly a major component of L. pneumophila intracellular infection.

scholarly journals PilZ Domain Protein FlgZ Mediates Cyclic Di-GMP-Dependent Swarming Motility Control in Pseudomonas aeruginosa

2016 ◽  
Vol 198 (13) ◽  
pp. 1837-1846 ◽  
Author(s):  
Amy E. Baker ◽  
Andreas Diepold ◽  
Sherry L. Kuchma ◽  
Jessie E. Scott ◽  
Dae Gon Ha ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Dependent Manner ◽  
Swarming Motility ◽  
Content Type ◽  
Bacterial Surface ◽  
Important Regulator ◽  
Green Fluorescent

ABSTRACTThe second messenger cyclic diguanylate (c-di-GMP) is an important regulator of motility in many bacterial species. InPseudomonas aeruginosa, elevated levels of c-di-GMP promote biofilm formation and repress flagellum-driven swarming motility. The rotation ofP. aeruginosa's polar flagellum is controlled by two distinct stator complexes, MotAB, which cannot support swarming motility, and MotCD, which promotes swarming motility. Here we show that when c-di-GMP levels are elevated, swarming motility is repressed by the PilZ domain-containing protein FlgZ and by Pel polysaccharide production. We demonstrate that FlgZ interacts specifically with the motility-promoting stator protein MotC in a c-di-GMP-dependent manner and that a functional green fluorescent protein (GFP)-FlgZ fusion protein shows significantly reduced polar localization in a strain lacking the MotCD stator. Our results establish FlgZ as a c-di-GMP receptor affecting swarming motility byP. aeruginosaand support a model wherein c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator.IMPORTANCEThe regulation of surface-associated motility plays an important role in bacterial surface colonization and biofilm formation. c-di-GMP signaling is a widespread means of controlling bacterial motility, and yet the mechanism whereby this signal controls surface-associated motility inP. aeruginosaremains poorly understood. Here we identify a PilZ domain-containing c-di-GMP effector protein that contributes to c-di-GMP-mediated repression of swarming motility byP. aeruginosa. We provide evidence that this effector, FlgZ, impacts swarming motility via its interactions with flagellar stator protein MotC. Thus, we propose a new mechanism for c-di-GMP-mediated regulation of motility for a bacterium with two flagellar stator sets, increasing our understanding of surface-associated behaviors, a key prerequisite to identifying ways to control the formation of biofilm communities.

scholarly journals Cytosolic Delivery of Multidomain Cargos by the N Terminus of Pasteurella multocida Toxin

2018 ◽  
Vol 86 (8) ◽  
Author(s):  
Nathan C. Clemons ◽  
Yuka Bannai ◽  
Elizabeth E. Haywood ◽  
Yiting Xu ◽  
James D. Buschbach ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Pasteurella Multocida ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Proteolytic Processing ◽  
Host Cells ◽  
Protein Targets ◽  
Content Type ◽  
Cytosolic Delivery ◽  
N Terminus

ABSTRACT The zoonotic pathogen Pasteurella multocida produces a 146-kDa modular toxin (PMT) that enters host cells and manipulates intracellular signaling through action on its Gα protein targets. The N terminus of PMT (PMT-N) mediates cellular uptake through receptor-mediated endocytosis, followed by the delivery of the C-terminal catalytic domain from acidic endosomes into the cytosol. The putative native cargo of PMT consists of a 710-residue polypeptide with three distinct modular subdomains (C1-C2-C3), where C1 contains a membrane localization domain (MLD), C2 has an as-yet-undefined function, and C3 catalyzes the deamidation of a specific active-site glutamine residue in Gα protein targets. However, whether the three cargo subdomains are delivered intact or undergo further proteolytic processing during or after translocation from the late endosome is unclear. Here, we demonstrate that PMT-N mediates the delivery of its native C-terminal cargo as a single polypeptide, corresponding to C1-C2-C3, including the MLD, with no evidence of cleavage between subdomains. We show that PMT-N also delivers nonnative green fluorescent protein (GFP) cargo into the cytosol, further supporting that the receptor-binding and translocation functions reside within PMT-N. Our findings further show that PMT-N can deliver C1-C2 alone but that the presence of C1-C2 is important for the cytosolic delivery of the catalytic C3 subdomain by PMT-N. In addition, we further refine the minimum C3 domain required for intracellular activity as comprising residues 1105 to 1278. These findings reinforce that PMT-N serves as the cytosolic delivery vehicle for C-terminal cargo and demonstrate that its native cargo is delivered intact as C1-C2-C3.

scholarly journals Identification and Verification of Ubiquitin-Activated Bacterial Phospholipases

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Maxx H. Tessmer ◽  
David M. Anderson ◽  
Adam M. Pickrum ◽  
Molly O. Riegert ◽  
Dara W. Frank
Keyword(s):  
Functional Properties ◽  
Mammalian Cells ◽  
Bacterial Species ◽  
Expression System ◽  
Host Cells ◽  
Achromobacter Xylosoxidans ◽  
Common Mechanism ◽  
Homologous Proteins ◽  
Content Type ◽  
Structural And Functional Properties

ABSTRACTExoU is a potent type III secretion system effector that is injected directly into mammalian cells by the opportunistic pathogenPseudomonas aeruginosa. As a ubiquitin-activated phospholipase A2(PLA2), ExoU exhibits cytotoxicity by cleaving membrane phospholipids, resulting in lysis of the host cells and inhibition of the innate immune response. Recently, ExoU has been established as a model protein for a group of ubiquitin-activated PLA2enzymes encoded by a variety of bacteria. Bioinformatic analyses of homologous proteins is a powerful approach that can complement and enhance the overall understanding of protein structure and function. To conduct homology studies, it is important to have efficient and effective tools to screen and to validate the putative homologs of interest. Here we make use of anEscherichia coli-based dual expression system to screen putative ubiquitin-activated PLA2enzymes from a variety of bacteria that are known to colonize humans and to cause human infections. The screen effectively identified multiple ubiquitin-activated phospholipases, which were validated using both biological and biochemical techniques. In this study, two new ExoU orthologs were identified and the ubiquitin activation of the rickettsial enzyme RP534 was verified. Conversely, ubiquitin was not found to regulate the activity of several other tested enzymes. Based on structural homology analyses, functional properties were predicted for AxoU, a unique member of the group expressed byAchromobacter xylosoxidans.IMPORTANCEBacterial phospholipases act as intracellular and extracellular enzymes promoting the destruction of phospholipid barriers and inflammation during infections. Identifying enzymes with a common mechanism of activation is an initial step in understanding structural and functional properties. These properties serve as critical information for the design of specific inhibitors to reduce enzymatic activity and ameliorate host cell death. In this study, we identify and verify cytotoxic PLA2enzymes from several bacterial pathogens. Similar to the founding member of the group, ExoU, these enzymes share the property of ubiquitin-mediated activation. The identification and validation of potential toxins from multiple bacterial species provide additional proteins from which to derive structural insights that could lead to paninhibitors useful for treating a variety of infections.

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