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Lipopolysaccharide of the Yersinia pseudotuberculosis Complex

Knirel, Yuriy A; Anisimov, Andrey P; Kislichkina, Angelina A; Kondakova, Anna N; Bystrova, Olga V; Vagaiskaya, Anastasia S; Shatalin, Konstantin Y; Shashkov, Alexander S; Dentovskaya, Svetlana V
Lipopolysaccharide (LPS), localized in the outer leaflet of the outer membrane, serves as the major surface component of the Gram-negative bacterial cell envelope responsible for the activation of the host's innate immune system. Variations of the LPS structure utilized by Gram-negative bacteria promote survival by providing resistance to components of the innate immune system and preventing recognition by TLR4. This review summarizes studies of the biosynthesis of Yersinia pseudotuberculosis complex LPSs, and the roles of their structural components in molecular mechanisms of yersiniae pathogenesis and immunogenesis.
PMID: 34680043
ISSN: 2218-273x
CID: 5068212

Inhibitors of bacterial H2S biogenesis targeting antibiotic resistance and tolerance

Shatalin, Konstantin; Nuthanakanti, Ashok; Kaushik, Abhishek; Shishov, Dmitry; Peselis, Alla; Shamovsky, Ilya; Pani, Bibhusita; Lechpammer, Mirna; Vasilyev, Nikita; Shatalina, Elena; Rebatchouk, Dmitri; Mironov, Alexander; Fedichev, Peter; Serganov, Alexander; Nudler, Evgeny
Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H2S)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of H2S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H2S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.
PMID: 34112687
ISSN: 1095-9203
CID: 4900242

CydDC functions as a cytoplasmic cystine reductase to sensitize Escherichia coli to oxidative stress and aminoglycosides

Mironov, Alexander; Seregina, Tatyana; Shatalin, Konstantin; Nagornykh, Maxim; Shakulov, Rustem; Nudler, Evgeny
l-cysteine is the source of all bacterial sulfurous biomolecules. However, the cytoplasmic level of l-cysteine must be tightly regulated due to its propensity to reduce iron and drive damaging Fenton chemistry. It has been proposed that in Escherichia coli the component of cytochrome bd-I terminal oxidase, the CydDC complex, shuttles excessive l-cysteine from the cytoplasm to the periplasm, thereby maintaining redox homeostasis. Here, we provide evidence for an alternative function of CydDC by demonstrating that the cydD phenotype, unlike that of the bona fide l-cysteine exporter eamA, parallels that of the l-cystine importer tcyP. Chromosomal induction of eamA, but not of cydDC, from a strong pLtetO-1 promoter (Ptet) leads to the increased level of extracellular l-cysteine, whereas induction of cydDC or tcyP causes the accumulation of cytoplasmic l-cysteine. Congruently, inactivation of cydD renders cells resistant to hydrogen peroxide and to aminoglycoside antibiotics. In contrast, induction of cydDC sensitizes cells to oxidative stress and aminoglycosides, which can be suppressed by eamA overexpression. Furthermore, inactivation of the ferric uptake regulator (fur) in Ptet-cydDC or Ptet-tcyP cells results in dramatic loss of survival, whereas catalase (katG) overexpression suppresses the hypersensitivity of both strains to H2O2 These results establish CydDC as a reducer of cytoplasmic cystine, as opposed to an l-cysteine exporter, and further elucidate a link between oxidative stress, antibiotic resistance, and sulfur metabolism.
PMID: 32900959
ISSN: 1091-6490
CID: 4622762

H2S: a Bacterial Defense Mechanism Against the Host Immune Response

Toliver-Kinsky, Tracy; Cui, Weihua; Törö, Gabor; Lee, Seung-Jin; Shatalin, Konstantin; Nudler, Evgeny; Szabo, Csaba
The biological mediator hydrogen sulfide (H2S) is produced by bacteria and has been shown to be cytoprotective against oxidative stress and to increase the sensitivity of various bacteria to a range of antibiotic drugs. Here we evaluated whether bacterial H2S provides resistance against the immune response, using two bacterial species that are common sources of nosocomial infections, Escherichia coli and Staphylococcus aureus Elevations in H2S increased the resistance of both species to immune-mediated killing. Clearance of infections with wild type and genetically H2S-deficient E. coli and S. aureus was compared in vitro and in mouse models of abdominal sepsis and burn wound infection. Also, inhibitors of H2S-producing enzymes were used to assess bacterial killing by leukocytes. We found that inhibition of bacterial H2S production can increase susceptibility of both bacterial species to rapid killing by immune cells and can improve bacterial clearance after severe burn, an injury that increases susceptibility to opportunistic infections. These findings support the role of H2S as a bacterial defense mechanism against the host response and implicate bacterial H2S inhibition as a potential therapeutic intervention in the prevention or treatment of infections.
PMID: 30323021
ISSN: 1098-5522
CID: 3369942

Mechanism of H2S-mediated protection against oxidative stress in Escherichia coli

Mironov, Alexander; Seregina, Tatyana; Nagornykh, Maxim; Luhachack, Lyly G; Korolkova, Natalya; Lopes, Liubov Errais; Kotova, Vera; Zavilgelsky, Gennady; Shakulov, Rustem; Shatalin, Konstantin; Nudler, Evgeny
Endogenous hydrogen sulfide (H2S) renders bacteria highly resistant to oxidative stress, but its mechanism remains poorly understood. Here, we report that 3-mercaptopyruvate sulfurtransferase (3MST) is the major source of endogenous H2S in Escherichia coli Cellular resistance to H2O2 strongly depends on the activity of mstA, a gene that encodes 3MST. Deletion of the ferric uptake regulator (Fur) renders mstA cells hypersensitive to H2O2 Conversely, induction of chromosomal mstA from a strong pLtetO-1 promoter (P tet -mstA) renders fur cells fully resistant to H2O2 Furthermore, the endogenous level of H2S is reduced in fur or sodA sodB cells but restored after the addition of an iron chelator dipyridyl. Using a highly sensitive reporter of the global response to DNA damage (SOS) and the TUNEL assay, we show that 3MST-derived H2S protects chromosomal DNA from oxidative damage. We also show that the induction of the CysB regulon in response to oxidative stress depends on 3MST, whereas the CysB-regulated l-cystine transporter, TcyP, plays the principle role in the 3MST-mediated generation of H2S. These findings led us to propose a model to explain the interplay between l-cysteine metabolism, H2S production, and oxidative stress, in which 3MST protects E. coli against oxidative stress via l-cysteine utilization and H2S-mediated sequestration of free iron necessary for the genotoxic Fenton reaction.
PMID: 28533366
ISSN: 1091-6490
CID: 2574702

S-nitrosylation of peroxiredoxin 1 contributes to viability of lung epithelial cells during Bacillus anthracis infection

Chung, Myung-Chul; Alem, Farhang; Hamer, Sarah G; Narayanan, Aarthi; Shatalin, Konstantin; Bailey, Charles; Nudler, Evgeny; Hakami, Ramin M
BACKGROUND: Using Bacillus anthracis as a model gram-positive bacterium, we investigated the effects of host protein S-nitrosylation during bacterial infection. B. anthracis possess a bacterial nitric oxide synthase (bNOS) that is important for its virulence and survival. However, the role of S-nitrosylation of host cell proteins during B. anthracis infection has not been determined. METHODS: Nitrosoproteomic analysis of human small airway epithelial cells (HSAECs) infected with toxigenic B. anthracis Sterne was performed, identifying peroxiredoxin 1 (Prx1) as one predominant target. Peroxidase activity of Prx during infection was measured using 2-Cys-Peroxiredoxin activity assay. Chaperone activity of S-nitrosylated Prx1 was measured by insulin aggregation assay, and analysis of formation of multimeric species using Native PAGE. Griess assay and DAF-2DA fluorescence assay were used to measure NO production. Cell viability was measured using the Alamar Blue assay and the ATPlite assay (Perkin Elmer). RESULTS: S-nitrosylation of Prx1 in Sterne-infected HSAECs leads to a decrease in its peroxidase activity while enhancing its chaperone function. Treatment with bNOS inhibitor, or infection with bNOS deletion strain, reduces S-nitrosylation of Prx1 and decreases host cell survival. Consistent with this, siRNA knockdown of Prx1 lowers bNOS-dependent protection of HSAEC viability. CONCLUSIONS: Anthrax infection results in S-nitrosylation of multiple host proteins, including Prx1. The nitrosylation-dependent decrease in peroxidase activity of Prx1 and increase in its chaperone activity is one factor contributing to enhancing infected cell viability. GENERAL SIGNIFICANCE: These results provide a new venue of mechanistic investigation for inhalational anthrax that could lead to novel and potentially effective countermeasures.
PMID: 27612662
ISSN: 0006-3002
CID: 2238822

Neonatal hypoxic ischemic brain injury upregulates cystathione b-synthase and mtor signaling pathway [Meeting Abstract]

Lechpammer, M; Tran, Y; Wintermark, P; Martinez-Cerdeno, V; Shatalin, K; Berman, R; Jensen, F; Nudler, E; Zagzag, D
Encephalopathy of prematurity (EOP) is a complex form of cerebral injury that occurs in the setting of either primary or secondary hypoxia-ischemia (HI) in the premature infant. In this study we have investigated in the rat model of EOP whether neonatal HI of the brain may in vivo alter the expression of cystathionine b-synthase (CBS) and the components of the mammalian target of rapamycin (mTOR) signaling pathway. We have performed unilateral carotid ligation and HI (UCL/HI) in Long-Evans rats at P6 and found by western blot and immunohistochemical analyses an increased expression of CBS in the white matter as early as 24 hours (P7) post procedure. CBS remained elevated through P21, and to the lesser extent in early adulthood (P40). All tested mTOR downstream targets (p70S6K and phospho-p70S6K & S6 and phospho-S6) were also overexpressed at the same time points in the UCL/HI rats compared to healthy controls. Importantly, this overexpression of mTOR components was not observed in rats treated with the mTOR inhibitor everolimus. Behavioral assays-open field locomotion test and three-chambered social choice test-performed on young adult rats (P35-37) following UCL/HI at P6, indicated hyperactive behavior and impaired preference for social novelty. Everolimus prevented both of these hall-marks of autism and restored behavioral patterns otherwise observed in healthy controls. Gait analysis has shown motor deficits in the hind paws of UCL/HI rats that were also significantly reduced by everolimus, indicating neurological protection/recovery of treated animals. Our results suggest that neonatal HI brain injury may cause its long term sequelae via upregulation of CBS and mTOR signaling pathway, as recently observed by us in patients with EOP, and propose this signaling cascade as a possible new molecular therapeutic target for this still untreatable cause of later life motor and autism-like behavioral deficits
ISSN: 1554-6578
CID: 3188392

Role of H2S and NO in Bacillus anthracis spore formation and virulence [Meeting Abstract]

Shatalin, K; Nudler, E
Many prokaryotic species generate hydrogen sulfide (H2S) and nitric oxide (NO) enzymatically, from cysteine and arginine, respectively, in their natural environments. Both gases are small freely diffusible signaling molecules that are known to be involved in numerous physiological and pathological processes in mammals. However the biochemistry and physiological role of these gases in bacteria remains largely unknown. We have shown that inactivation of H2S producing enzymes (cystathionine beta-synthase, cystathionine gamma lyase, or 3-mercaptopyruvate sulfurtransferase) and NO-synthase in several Gram (+) and Gram (-) bacteria render them highly sensitive to different classes of antibiotics (Gusarov et al., Science 325 (2009) 1380-1384; Shatalin et al. Science 334 (2011) 986-990). We also presented evidence that Bacillus anthracis-derived NO is critical at the early stage of infection (Shatalin et al. PNAS 105 (2008) 1009-1013). Here we show that: (1) cbs/cse and nos mutations change Bacilli global gene transcription profile; (2) apore formation process in cbs/cse and nos mutants ofB. anthracis is affected; (3) virulence of cbs/cse and nos mutants of B. anthracis is diminished. These results demonstrate that bacterial H2S and NO are an important virulence factors, and that enzymes generated these gases may serve as an attractive target for antimicrobial therapy
ISSN: 1089-8603
CID: 1154012

H2S: a universal defense against antibiotics in bacteria

Shatalin, Konstantin; Shatalina, Elena; Mironov, Alexander; Nudler, Evgeny
Many prokaryotic species generate hydrogen sulfide (H(2)S) in their natural environments. However, the biochemistry and physiological role of this gas in nonsulfur bacteria remain largely unknown. Here we demonstrate that inactivation of putative cystathionine beta-synthase, cystathionine gamma-lyase, or 3-mercaptopyruvate sulfurtransferase in Bacillus anthracis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli suppresses H(2)S production, rendering these pathogens highly sensitive to a multitude of antibiotics. Exogenous H(2)S suppresses this effect. Moreover, in bacteria that normally produce H(2)S and nitric oxide, these two gases act synergistically to sustain growth. The mechanism of gas-mediated antibiotic resistance relies on mitigation of oxidative stress imposed by antibiotics
PMID: 22096201
ISSN: 1095-9203
CID: 141709

Linking RNA Polymerase Backtracking to Genome Instability in E. coli

Dutta, Dipak; Shatalin, Konstantin; Epshtein, Vitaly; Gottesman, Max E; Nudler, Evgeny
Frequent codirectional collisions between the replisome and RNA polymerase (RNAP) are inevitable because the rate of replication is much faster than that of transcription. Here we show that, in E. coli, the outcome of such collisions depends on the productive state of transcription elongation complexes (ECs). Codirectional collisions with backtracked (arrested) ECs lead to DNA double-strand breaks (DSBs), whereas head-on collisions do not. A mechanistic model is proposed to explain backtracking-mediated DSBs. We further show that bacteria employ various strategies to avoid replisome collisions with backtracked RNAP, the most general of which is translation that prevents RNAP backtracking. If translation is abrogated, DSBs are suppressed by elongation factors that either prevent backtracking or reactivate backtracked ECs. Finally, termination factors also contribute to genomic stability by removing arrested ECs. Our results establish RNAP backtracking as the intrinsic hazard to chromosomal integrity and implicate active ribosomes and other anti-backtracking mechanisms in genome maintenance
PMID: 21854980
ISSN: 1097-4172
CID: 136948