Stephanie R. Shames

Michigan State University

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B. Brett Finlay

University of British Columbia

Tshegofatso Ngwaga

Kansas State University

Deepika Chauhan

University of Missouri

Ashley M. Joseph

Kansas State University

Wanyin Deng

University of British Columbia

Matthew A. Croxen

University of Alberta

Ramasamy Perumal

Fort Hays State University

Leela E. Noronha

Center for Grain and Animal Health Research

Dmitriy Smolensky

Center for Grain and Animal Health Research

Sarah Cox

University of Cambridge

Cooperative Institutions

University of British Columbia

Canada's Michael Smith Genome Sciences Centre

Kansas State University

Upsher-Smith Laboratories (United Kingdom)

Yale University

Bipar

British Columbia Children's Hospital

Louisiana State University Health Sciences Center Shreveport

Louisiana State University Health Sciences Center New Orleans

Tokyo University of Pharmacy and Life Sciences

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High-polyphenol extracts from Sorghum bicolor attenuate replication of Legionella pneumophila within RAW 264.7 macrophages

FEMS Microbiology Letters (2020)

Aubrey K Gilchrist Dmitriy Smolensky Tshegofatso Ngwaga Deepika Chauhan Sarah Cox

ABSTRACT Polyphenols derived from a variety of plants have demonstrated antimicrobial activity against diverse microbial pathogens. Legionella pneumophila is an intracellular bacterial pathogen that opportunistically causes a severe inflammatory pneumonia in humans, called Legionnaires’ Disease, via replication within macrophages. Previous studies demonstrated that tea polyphenols attenuate L. pneumophila intracellular replication within mouse macrophages via increased tumor necrosis factor (TNF) production. Sorghum bicolor is a sustainable cereal crop that thrives in arid environments and is well-suited to continued production in warming climates. Sorghum polyphenols have anticancer and antioxidant properties, but their antimicrobial activity has not been evaluated. Here, we investigated the impact of sorghum polyphenols on L. pneumophila intracellular replication within RAW 264.7 mouse macrophages. Sorghum high-polyphenol extract (HPE) attenuated L. pneumophila intracellular replication in a dose-dependent manner but did not impair either bacterial replication in rich media or macrophage viability. Moreover, HPE treatment enhanced both TNF and IL-6 secretion from L. pneumophila infected macrophages. Thus, polyphenols derived from sorghum enhance macrophage restriction of L. pneumophila, likely via increased pro-inflammatory cytokine production. This work reveals commonalities between plant polyphenol-mediated antimicrobial activity and provides a foundation for future evaluation of sorghum as an antimicrobial agent.

Legionella

10.1093/femsle/fnaa053

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Citations (9)

Bacterial effector interplay: a new way to view effector function

Trends in Microbiology (2012)

Stephanie R. Shames B. Brett Finlay

10.1016/j.tim.2012.02.007

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Citations (50)

Quantitative Mass Spectrometry Identifies Novel Host Binding Partners for Pathogenic Escherichia coli Type III Secretion System Effectors

Journal of Proteome Research (2016)

Robyn J. Law Hong T. Law Joshua Scurll Roland W. Scholz Andrew S. Santos

Enteropathogenic and enterohemorrhagic Escherichia coli cause enteric diseases resulting in significant morbidity and mortality worldwide. These pathogens remain extracellular and translocate a set of type III secreted effector proteins into host cells to promote bacterial virulence. Effectors manipulate host cell pathways to facilitate infection by interacting with a variety of host targets, yet the binding partners and mechanism of action of many effectors remain elusive. We performed a mass spectrometry screen to identify host targets for a library of effectors. We found five known effector targets and discovered four novel interactions. Interestingly, we identified multiple effectors that interacted with the microtubule associated protein, ensconsin. Using co-immunoprecipitations, we confirmed that NleB1 and EspL interacted with ensconsin in a region that corresponded to its microtubule binding domain. Ensconsin is an essential cofactor of kinesin-1 that is required for intracellular trafficking, and we demonstrated that intracellular trafficking was severely disrupted during wild type EPEC infections but not during infections with ΔnleB1 or ΔespL mutants. Our findings demonstrate the efficacy of quantitative proteomics for identifying effector–host protein interactions and suggest that vesicular trafficking is a crucial cellular process that may be targeted by NleB1 and EspL through their interaction with ensconsin.

Intracellular parasite

10.1021/acs.jproteome.6b00074

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Carbapenem-Resistant Enterobacter hormaechei Uses Mucus Metabolism to Facilitate Gastrointestinal Colonization

bioRxiv (Cold Spring Harbor Laboratory) (2024)

Ritam Sinha Elizabeth N. Ottosen Tshegofatso Ngwaga Stephanie R. Shames Victor J. DiRita

The emergence and global spread of carbapenem-resistant

10.1101/2024.09.25.615021

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Intrabacterial Regulation of a Cytotoxic Effector by Its Cognate Metaeffector Promotes Legionella pneumophila Virulence

mSphere (2023)

Deepika Chauhan Ashley M. Joseph Stephanie R. Shames

Legionella pneumophila replicates within phagocytic host cells using hundreds of effector protein virulence factors, which canonically subvert the function of host proteins and pathways. L. pneumophila encodes a unique family of translocated effectors called metaeffectors, which bind and regulate the function of a cognate effector in host cells. The metaeffector MesI promotes L. pneumophila virulence by regulating the cytotoxic effector SidI; however, the MesI regulatory mechanism is poorly understood.

Legionella

10.1128/msphere.00552-22

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The Legionella pneumophila Metaeffector Lpg2505 (MesI) Regulates SidI-Mediated Translation Inhibition and Novel Glycosyl Hydrolase Activity

Infection and Immunity (2020)

Ashley M. Joseph Adrienne E. Pohl Theodore J. Ball Troy G. Abram David K. Johnson

Legionella pneumophila, the etiological agent of Legionnaires' disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

Legionella

Eukaryotic translation

10.1128/iai.00853-19

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The pathogenic Escherichia coli type III secreted protease NleC degrades the host acetyltransferase p300

Cellular Microbiology (2011)

Stephanie R. Shames Amit P. Bhavsar Matthew A. Croxen Robyn J. Law Shinghung Mak

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively) are attaching and effacing bacterial pathogens that cause devastating diarrhoeal disease worldwide. These pathogens depend on a type III secretion system, which functions as a molecular syringe to translocate bacterial effector proteins directly into infected host cells. One of these effectors, NleC, was recently described as a zinc metalloprotease that targets NF-κB Rel-A (p65) and thus contributes to dampening of inflammatory signalling during EPEC and EHEC infection. We have identified the acetyltransferase p300 as an additional target of NleC. Several biochemical techniques were employed to demonstrate specific binding of p300 by NleC. We also show that NleC causes decreased abundance of p300 in cellular nuclei and that the metalloprotease domain of NleC is responsible for this phenotype. Furthermore, we demonstrate that overexpression of p300 can antagonize repression of IL-8 secretion by EPEC and that siRNA knock-down of p300 dampens IL-8 secretion by EPEC ΔnleC-infected cells. We have therefore identified a second target of NleC and provided the first example of a bacterial virulence factor targeting the acetyltransferase p300.

Enteropathogenic Escherichia coli

Virulence factor

10.1111/j.1462-5822.2011.01640.x

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Citations (56)

High Polyphenol Sorghum Extract Attenuates Th2 cytokine Profile and Reduces Growth of Legionella in Raw 264.7 Mouse Macrophage Cell Line

Current Developments in Nutrition (2020)

Dmitriy Smolensky Sarah Cox Stephanie R. Shames Leela E. Noronha Weiqun Wang

1) To evaluate the changes in inflammatory response induced by sorghum polyphenols in activated macrophages and 2) to evaluate possible efficacy of sorghum polyphenols on an opportunistic intracellular pathogen, Legionella pneumophila (LP). Raw 265.7 cells mouse macrophage cells were treated with sorghum phenolic extract (SPE) under control and activating conditions to evaluate the role of SPE in inflammatory and anti-inflammatory processes. Study measured: nitric oxide production in the supernatant, mRNA using qPCR array of 84 genes in activated macrophages. Morphological changes were observed, and LC3 protein expression was measured to test for autophagy using western blot. NF-kB and STAT3 nuclear translocation was measured using a fractionization kit, followed by western blot. The replication of LP was measured within RAW 264.7 cells and in vitro (in media without cell presence). Cytotoxicity assay and a western blot apoptosis marker caspase-3 were used to evaluate the cytotoxicity of SPE on RAW 264.7 cells. Because LP reproduction within cells is greatly attenuated in the presence of Tumor Necrosis Factor (TNF), LP replication was measured under the presence of TNF neutralizing antibodies. SPE decreased nitric oxide production in activated LPS/IFNΥ macrophages although not significantly. SPE attenuated Th2 cytokine response in LPS/IFNΥ activated macrophages by decreasing expression of IL-6 and IL-10 while not changing expression of other inflammatory cytokines. Quantitative PCR data confirmed that genes in the IL-10 and IL-6 pathway were downregulated by cotreatment of SPE and LPS/IFNΥ when compared to LPS/IFNΥ alone. Morphological changes observed exhibited formation of large vacuole like structures. Analysis of LC3 confirmed that autophagy was increased in activated cells treated with SPE. Nuclear fractionization confirmed that STAT3 signaling was attenuated by SPE in activated macrophages. SPE significantly reduced the replication of LP in macrophage cells but not in vitro. The attenuation of LP grown in RAW 264.7 cells was independent of TNF presence. This data suggests that sorghum phenolic compounds may have potential pharmaceutical/nutritional uses to combat intracellular pathogens. All funding was provided by the United States Department of Agriculture.

10.1093/cdn/nzaa068_022

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Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

Proceedings of the National Academy of Sciences (2017)

Stephanie R. Shames Luying Liu James C. Havey Whitman B. Schofield Andrew L. Goodman

Significance The contribution of individual effectors to Legionella pneumophila virulence has not been systematically examined. This study employed a parallel high-throughput transposon insertion sequencing technique called INSeq to probe the L. pneumophila effector repertoire and identified multiple effectors that contribute to virulence in several host organisms, including an animal model of Legionnaires’ disease. Importantly, this study demonstrates that effector proteins contribute to host virulence both positively and negatively by controlling intracellular replication and influencing host immune responses, which demonstrates that the subtle alterations in the effector repertoire of a single L. pneumophila strain can greatly impact host pathogenicity.

Legionella

10.1073/pnas.1708553114

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Citations (87)

The Genus Legionella

CRC Press eBooks (2021)

Ashley M. Joseph Stephanie R. Shames

Members of the genus Legionella are Gram-negative, aerobic bacilli that belong to the gamma-subgroup of proteobacteria. Legionella are facultative, intracellular bacteria that are ubiquitously found in freshwater ponds and streams where they colonize phagocytic protozoa. The relevance of these organisms to human health was first recognized following the eponymous 1976 outbreak at a meeting of the American Legion in Philadelphia. Subsequent outbreaks of Legionella infection have been traced to several types of man-made water reservoirs, including cooling towers, industrial air conditioners, hot water systems and fountains. The diseases resulting from exposure to Legionella, collectively termed legionellosis, include Legionnaires' disease and Pontiac fever. Legionnaires' disease is a severe infection of the respiratory tract that is fatal in approximately 10% of cases. Legionella infection is one of the most common causes of community-acquired pneumonia (CAP) and is second only to bacteremic pneumococcal pneumonia in causing severe CAP that requires treatment in intensive care.

Legionella

10.1201/9781003099277-33

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