Biology subjects

Shames, S. R.

Publications and source records attributed to Shames, S. R..

7 recordsLinked to original sources

bioRxiv2025

Crystal structure of the Legionella pneumophila effector SidL (Lpg0437) in complex with its metaeffector LegA11 (Lpg0436)

Legionella pneumophila is an opportunistic human pathogen that causes atypical pneumonia called Legionnaires Disease. To replicate within host cells, L. pneumophila injects up to 330 effector proteins into the host cytosol via a Dot/Icm type IV secretion system. Several effectors, called metaeffectors, regulate the activity of other effectors within infected host cells through direct protein-protein interactions. LegA11 (AnkJ/Lpg0436) has been identified as a putative metaeffector of SidL (Ceg14/Lpg0437), one of eight L. pneumophila effectors that inhibit host mRNA translation. LegA11 binds and suppresses SidL enzymatic activity, but the molecular basis of this regulation and impact on mRNA translation are unknown. Here, we present the crystal structure of SidL in complex with LegA11 to a resolution of 2.4 [A], revealing a high-affinity 1:1 complex with an extensive interaction interface of [~]2300 [A]{superscript 2}. Using isothermal titration calorimetry, we determined a dissociation constant of 1.8 nM. In vitro translation assays demonstrate that SidL inhibits mRNA translation, and this activity is completely suppressed by LegA11. Mutagenesis of key interface residues in LegA11 disrupts complex formation and abolishes its metaeffector activity, confirming that LegA11 regulates SidL through direct protein-protein interaction. These findings establish LegA11 as a bona fide metaeffector that contributes to suppression of host mRNA translation by L. pneumophila.

microbiology↗

bioRxiv2024

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

The emergence and global spread of carbapenem-resistant Enterobacter cloacae complex species presents a pressing public health challenge. Carbapenem-resistant Enterobacter species cause a wide variety of infections, including septic shock fatalities in newborns and immunocompromised adults. The intestine may be a major reservoir for these resistant strains, either by facilitating contamination of fomites and transfer to susceptible individuals, or through translocation from the gut to the bloodstream. For this reason, we sought to establish a neonatal mouse model to investigate the mechanisms underpinning gut colonization by carbapenem-resistant Enterobacter hormaechei. We describe a new mouse model to study gut colonization by Enterobacter species, leading to vital insights into the adaptation of carbapenem-resistant E. hormaechei to the gut environment during the early stages of intestinal colonization. We observed successful colonization and proliferation of E. hormaechei in the five-day old infant mouse gut, with primary localization to the colon following oral inoculation. We also uncovered evidence that E. hormaechei uses mucus as a carbon source during colonization of the colon. Our findings underscore the importance of oxygen-dependent metabolic pathways, including the pyruvate dehydrogenase complex, and N-acetyl-D-glucosamine metabolism, in gut colonization and proliferation, which aligns with previous human studies. These insights are essential for developing novel therapeutic strategies that can serve as decolonization therapies in at-risk populations. ImportanceBloodstream infections caused by Enterobacter species pose a significant clinical threat. The intestine acts as the primary site for colonization and serves as a reservoir for infection. To combat this pathogen, it is crucial to understand how carbapenem-resistant Enterobacter species colonize the gut, as such knowledge can pave the way for alternative therapeutic targets. In this study, we developed a novel neonatal mouse model for gastrointestinal colonization by Enterobacter species and discovered that mucus plays a key role as a carbon source during colonization. Additionally, we identified two mucus catabolism pathways that contribute to intestinal colonization by carbapenem-resistant E. hormaechei. This new mouse model offers valuable insights into host-pathogen interactions and helps identify critical gastrointestinal fitness factors of Enterobacter, potentially guiding the development of vaccines and alternative therapeutic strategies to minimize intestinal carriage in patient populations at risk for infection with Enterobacter species.

microbiology↗

bioRxiv2022

Intrabacterial regulation of a cytotoxic effector by its cognate metaeffector promotes Legionella pneumophila virulence

Legionella pneumophila is a natural pathogen of unicellular protozoa that can opportunistically infect macrophages and cause Legionnaires Disease. Intracellular replication is driven by hundreds of bacterial effector proteins that are translocated into infected host cells by a Dot/Icm type IV secretion system. L. pneumophila effectors are temporally regulated in part by a unique family of translocated regulatory effectors, termed metaeffectors, which bind and modulate the function of a cognate effector in host cells. We discovered that regulation of the cytotoxic effector SidI by its metaeffector, MesI, is critical for L. pneumophila virulence in natural and opportunistic hosts. MesI binds and negatively regulates SidI activity in vitro but how dysregulation of SidI impairs L. pneumophila intracellular replication is unclear. Using a chromosomally-encoded inducible expression system, we discovered that dysregulation of SidI, via loss of MesI, was toxic to L. pneumophila. SidI enzymatic activity was required for toxicity since L. pneumophila growth was unaffected by induced expression of a catalytically inactive sidI allele. We found MesI translocation into host cells was dispensable for intracellular replication and that MesI-deficient bacteria were rapidly degraded within host cells. Together, our data suggest a unique role for intrabacterial effector regulation by a translocated metaeffector in L. pneumophila virulence. ImportanceLegionella 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. We discovered a unique intrabacterial role for MesI in L. pneumophila virulence. When uncoupled from MesI, SidI was toxic to L. pneumophila in vitro and triggered robust bacterial degradation in host cells. Importantly, translocation of MesI was dispensable for intracellular replication, demonstrating that intrabacterial regulation of SidI contributes to L. pneumophila virulence. These data show a unique and important role for MesI regulation of SidI within the bacterium, which challenges the dogma that effectors function exclusively within host cells.

microbiology↗

bioRxiv2022

Effector-mediated subversion of proteasome activator (PA)28αβ enhances lysosomal targeting of Legionella pneumophila within cytokine-activated macrophages

Legionella pneumophila is a natural pathogen of amoebae that causes Legionnaires Disease in immunocompromised individuals via replication within macrophages. L. pneumophila virulence and intracellular replication hinges on hundreds of Dot/Icm-translocated effector proteins, which are essential for biogenesis of the replication permissive Legionella-containing vacuole (LCV). However, effector activity can also enhance mammalian host defense via effector-triggered immunity. The L. pneumophila effector LegC4 is important for virulence in amoebae but enhances host defense against L. pneumophila in the mouse lung and, uniquely, within macrophages activated with either tumor necrosis factor (TNF) or interferon (IFN)-{gamma}. The mechanism by which LegC4 potentiates cytokine-mediated host defense in macrophages is unknown. Here, we found that LegC4 enhances cytokine-mediated phagolysosomal fusion with Legionella-containing vacuole (LCV) and binds host proteasome activator (PA)28, which forms a heterooligomer with PA28{beta} to facilitate ubiquitin-independent proteasomal degradation of oxidant-damaged (carbonylated) proteins. We found that oxidative stress was sustained in the presence of LegC4 and that the LegC4 restriction phenotype was relieved in PA28{beta}-deficient macrophages and in the lungs of mice in vivo. Our data also show that oxidative stress is sufficient for LegC4-mediated restriction in macrophages producing PA28{beta}. PA28{beta} has been traditionally associated with antigen presentation; however, our data support a novel mechanism whereby effector-mediated subversion of PA28{beta} enhances cell-autonomous host defense against L. pneumophila under inflammatory and oxidative stress conditions. This work provides a solid foundation to evaluate induced proteasome regulators as mediators of innate immunity. Author SummaryPro-inflammatory cytokines induce antimicrobial host defense pathways within macrophages to control intracellular pathogens. We discovered that the Legionella pneumophila effector protein LegC4 potentiates pathogen clearance within cytokine-activated macrophages. Here, we show a central role for host proteasome activator (PA)28{beta} in LegC4 restriction. PA28{beta} is upregulated by cytokine signaling and under oxidative stress conditions to facilitate proteasomal degradation of oxidant-damaged proteins. We found that LegC4 binds PA28{beta} and that LegC4 restriction was lost in PA28{beta}-deficient (Psme1/2-/-) macrophages and a mouse model of Legionnaires Disease in vivo. Furthermore, oxidative stress was sustained in the presence of LegC4 and was sufficient for LegC4 restriction in PA28{beta}-producing cells macrophages. Finally, we found that L. pneumophila replication was attenuated within PA28{beta}-deficient macrophages irrespective of LegC4. These findings support a model whereby subversion of host proteostasis machinery triggers cell-autonomous host defense within macrophages under inflammatory and oxidative stress conditions.

microbiology↗

bioRxiv2019

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

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 amplification of tumor necrosis factor (TNF) production. Sorghum bicolor is a sustainable and resilient cereal crop that thrives in arid environments and is well suited to continued production in warming climates. Polyphenols derived from sorghum 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. We discovered that sorghum high-polyphenol extract (HPE) treatment attenuates L. pneumophila intracellular replication in a dose-dependent manner. Sorghum HPE did not impair bacterial replication in vitro or impact macrophage viability. Moreover, in contrast to tea polyphenols, HPE treatment impaired TNF secretion from infected macrophages. Thus, polyphenols derived from sorghum enhance macrophage restriction of L. pneumophila by a novel mechanism. This work provides a foundation for the use of sorghum as an antimicrobial agent.

microbiology↗

bioRxiv2019

The Legionella pneumophila metaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity.

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 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 SusF is not mutually exclusive and these proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyltransferase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505, SusF (suppressor of SidI function). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

microbiology↗

bioRxiv2018

The Dot/Icm-translocated effector LegC4 potentiates cytokine-mediated restriction of Legionella within accidental hosts.

Legionella pneumophila is ubiquitous in freshwater environments where it replicates within unicellular protozoa. However, L. pneumophila is also an accidental human pathogen that can cause Legionnaires Disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila utilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction of L. pneumophila. We found previously that the effector LegC4 is important for L. pneumophila replication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires Disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuates L. pneumophila replication. We found that LegC4 enhanced restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon (IFN)-{gamma}. Specifically, TNF-mediated signaling was required for LegC4-mediated attenuation of L. pneumophila replication within macrophages. In addition, expression of legC4 was sufficient to restrict L. longbeachae replication within TNF- or IFN-{gamma}-activated macrophages. Thus, this study demonstrates that LegC4 contributes to L. pneumophila clearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.\n\nImportanceLegionella are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controls Legionella infection due in part to rapid and robust production of pro-inflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances pro-inflammatory host restriction of Legionella from macrophages. These data suggest that LegC4 may augment pro-inflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens.

microbiology↗