Bacterial Persisters and Infection: Past, Present, and Progressing.

Persisters are nongrowing, transiently antibiotic-tolerant bacteria within a clonal population of otherwise susceptible cells. Their formation is triggered by environmental cues and involves the main bacterial stress response pathways that allow persisters to survive many harsh conditions, including antibiotic exposure. During infection, bacterial pathogens are exposed to a vast array of stresses in the host and form nongrowing persisters that survive both antibiotics and host immune responses, thereby most likely contributing to the relapse of many infections. While antibiotic persisters have been extensively studied over the last decade, the bulk of the work has focused on how these bacteria survive exposure to drugs in vitro. The ability of persisters to survive their interaction with a host is important yet underinvestigated. In order to tackle the problem of persistence of infections that contribute to the worldwide antibiotic resistance crisis, efforts should be made by scientific communities to understand and merge these two fields of research: antibiotic persisters and host-pathogen interactions. Here we give an overview of the history of the field of antibiotic persistence, report evidence for the importance of persisters in infection, and highlight studies that bridge the two areas.

Dual-Activity Fluoroquinolone-Transportan 10 Conjugates Offer Alternative Leukemia Therapy during Hematopoietic Cell Transplantation.

Hematopoietic cell transplantation (HCT) is often considered a last resort leukemia treatment, fraught with limited success due to microbial infections, a leading cause of mortality in leukemia patients. To address this critical issue, we explored a novel approach by synthesizing antileukemic agents containing antibacterial substances. This innovative strategy involves conjugating fluoroquinolone antibiotics, such as ciprofloxacin (CIP) or levofloxacin (LVX), with the cell-penetrating peptide transportan 10 (TP10). Here, we demonstrate that the resultant compounds display promising biologic activities in preclinical studies. These novel conjugates not only exhibit potent antimicrobial effects but are also selective against leukemia cells. The cytotoxic mechanism involves rapid disruption of cell membrane asymmetry leading to membrane damage. Importantly, these conjugates penetrated mammalian cells, accumulating within the nuclear membrane without significant effect on cellular architecture or mitochondrial function. Molecular simulations elucidated the aggregation tendencies of TP10 conjugates within lipid bilayers, resulting in membrane disruption and permeabilization. Moreover, mass spectrometry analysis confirmed efficient reduction of disulfide bonds within TP10 conjugates, facilitating release and activation of the fluoroquinolone derivatives. Intriguingly, these compounds inhibited human topoisomerases, setting them apart from traditional fluoroquinolones. Remarkably, TP10 conjugates generated lower intracellular levels of reactive oxygen species compared with CIP and LVX. The combination of antibacterial and antileukemic properties, coupled with selective cytostatic effects and minimal toxicity toward healthy cells, positions TP10 derivatives as promising candidates for innovative therapeutic approaches in the context of antileukemic HCT. This study highlights their potential in search of more effective leukemia treatments. SIGNIFICANCE STATEMENT: Fluoroquinolones are commonly used antibiotics, while transportan 10 (TP10) is a cell-penetrating peptide (CPP) with anticancer properties. In HCT, microbial infections are the primary cause of illness and death. Combining TP10 with fluoroquinolones enhanced their effects on different cell types. The dual pharmacological action of these conjugates offers a promising proof-of-concept solution for leukemic patients undergoing HCT. Strategically designed therapeutics, incorporating CPPs with antibacterial properties, have the potential to reduce microbial infections in the treatment of malignancies.

Auxiliary interfaces support the evolution of specific toxin-antitoxin pairing.

Toxin-antitoxin (TA) systems are a large family of genes implicated in the regulation of bacterial growth and its arrest in response to attacks. These systems encode nonsecreted toxins and antitoxins that specifically pair, even when present in several paralogous copies per genome. Salmonella enterica serovar Typhimurium contains three paralogous TacAT systems that block bacterial translation. We determined the crystal structures of the three TacAT complexes to understand the structural basis of specific TA neutralization and the evolution of such specific pairing. In the present study, we show that alteration of a discrete structural add-on element on the toxin drives specific recognition by their cognate antitoxin underpinning insulation of the three pairs. Similar to other TA families, the region supporting TA-specific pairing is key to neutralization. Our work reveals that additional TA interfaces beside the main neutralization interface increase the safe space for evolution of pairing specificity.

Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism.

Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A 'gatekeeper' complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region's structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.

Effective Drug Concentration and Selectivity Depends on Fraction of Primitive Cells.

Poor efficiency of chemotherapeutics in the eradication of Cancer Stem Cells (CSCs) has been driving the search for more active and specific compounds. In this work, we show how cell density-dependent stage culture profiles can be used in drug development workflows to achieve more robust drug activity (IC50 and EC50) results. Using flow cytometry and light microscopy, we characterized the cytological stage profiles of the HL-60-, A-549-, and HEK-293-derived sublines with a focus on their primitive cell content. We then used a range of cytotoxic substances-C-123, bortezomib, idarubicin, C-1305, doxorubicin, DMSO, and ethanol-to highlight typical density-related issues accompanying drug activity determination. We also showed that drug EC50 and selectivity indices normalized to primitive cell content are more accurate activity measurements. We tested our approach by calculating the corrected selectivity index of a novel chemotherapeutic candidate, C-123. Overall, our study highlights the usefulness of accounting for primitive cell fractions in the assessment of drug efficiency.

Inhibition of Nuclear Transport of NF-ĸB p65 by the Salmonella Type III Secretion System Effector SpvD.

Salmonella enterica replicates in macrophages through the action of effector proteins translocated across the vacuolar membrane by a type III secretion system (T3SS). Here we show that the SPI-2 T3SS effector SpvD suppresses proinflammatory immune responses. SpvD prevented activation of an NF-ĸB-dependent promoter and caused nuclear accumulation of importin-α, which is required for nuclear import of p65. SpvD interacted specifically with the exportin Xpo2, which mediates nuclear-cytoplasmic recycling of importins. We propose that interaction between SpvD and Xpo2 disrupts the normal recycling of importin-α from the nucleus, leading to a defect in nuclear translocation of p65 and inhibition of activation of NF-ĸB regulated promoters. SpvD down-regulated pro-inflammatory responses and contributed to systemic growth of bacteria in mice. This work shows that a bacterial pathogen can manipulate host cell immune responses by interfering with the nuclear transport machinery.

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl.

The Salmonella Effector SpvD Is a Cysteine Hydrolase with a Serovar-specific Polymorphism Influencing Catalytic Activity, Suppression of Immune Responses, and Bacterial Virulence.

Many bacterial pathogens secrete virulence (effector) proteins that interfere with immune signaling in their host. SpvD is a Salmonella enterica effector protein that we previously demonstrated to negatively regulate the NF-κB signaling pathway and promote virulence of S. enterica serovar Typhimurium in mice. To shed light on the mechanistic basis for these observations, we determined the crystal structure of SpvD and show that it adopts a papain-like fold with a characteristic cysteine-histidine-aspartate catalytic triad comprising Cys-73, His-162, and Asp-182. SpvD possessed an in vitro deconjugative activity on aminoluciferin-linked peptide and protein substrates in vitro A C73A mutation abolished SpvD activity, demonstrating that an intact catalytic triad is required for its function. Taken together, these results strongly suggest that SpvD is a cysteine protease. The amino acid sequence of SpvD is highly conserved across different S. enterica serovars, but residue 161, located close to the catalytic triad, is variable, with serovar Typhimurium SpvD having an arginine and serovar Enteritidis a glycine at this position. This variation affected hydrolytic activity of the enzyme on artificial substrates and can be explained by substrate accessibility to the active site. Interestingly, the SpvDG161 variant more potently inhibited NF-κB-mediated immune responses in cells in vitro and increased virulence of serovar Typhimurium in mice. In summary, our results explain the biochemical basis for the effect of virulence protein SpvD and demonstrate that a single amino acid polymorphism can affect the overall virulence of a bacterial pathogen in its host.

Molecular stripping underpins derepression of a toxin-antitoxin system.

Transcription factors control gene expression; among these, transcriptional repressors must liberate the promoter for derepression to occur. Toxin-antitoxin (TA) modules are bacterial elements that autoregulate their transcription by binding the promoter in a T:A ratio-dependent manner, known as conditional cooperativity. The molecular basis of how excess toxin triggers derepression has remained elusive, largely because monitoring the rearrangement of promoter-repressor complexes, which underpin derepression, is challenging. Here, we dissect the autoregulation of the Salmonella enterica tacAT3 module. Using a combination of assays targeting DNA binding and promoter activity, as well as structural characterization, we determine the essential TA and DNA elements required to control transcription, and we reconstitute a repression-to-derepression path. We demonstrate that excess toxin triggers molecular stripping of the repressor complex off the DNA through multiple allosteric changes causing DNA distortion and ultimately leading to derepression. Thus, our work provides important insight into the mechanisms underlying conditional cooperativity.

Stapled Phd Peptides Inhibit Doc Toxin Induced Growth Arrest in Salmonella.

Bacterial toxin inhibition is a promising approach to overcoming antibiotic failure. InSalmonella, knockout of the toxin Doc has been shown to significantly reduce the formation of antibiotic-tolerant persisters. Doc is a kinase that is inhibited in nontolerant cells by its cognate antitoxin, Phd. In this work, we have developed first-in-class stapled peptide antitoxin mimetics based on the Doc inhibitory sequence of Phd. After making a series of substitutions to improve bacterial uptake, we identified a lead stapled Phd peptide that is able to counteract Doc toxicity in Salmonella. This provides an exciting starting point for the further development of therapeutic peptides capable of reducing antibiotic persistence in pathogenic bacteria.

Characterization of the Key Determinants of Phd Antitoxin Mediated Doc Toxin Inactivation in Salmonella.

In the search for novel antimicrobial therapeutics, toxin-antitoxin (TA) modules are promising yet underexplored targets for overcoming antibiotic failure. The bacterial toxin Doc has been associated with the persistence of Salmonella in macrophages, enabling its survival upon antibiotic exposure. After developing a novel method to produce the recombinant toxin, we have used antitoxin-mimicking peptides to thoroughly investigate the mechanism by which its cognate antitoxin Phd neutralizes the activity of Doc. We reveal insights into the molecular detail of the Phd-Doc relationship and discriminate antitoxin residues that stabilize the TA complex from those essential for inhibiting the activity of the toxin. Coexpression of Doc and antitoxin peptides in Salmonella was able to counteract the activity of the toxin, confirming our in vitro results with equivalent sequences. Our findings provide key principles for the development of chemical tools to study and therapeutically interrogate this important class of protein-protein interactions.

A Bacterial Cell-Based Assay To Study SARS-CoV-2 Protein-Protein Interactions.

Methods for detecting and dissecting the interactions of virally encoded proteins are essential for probing basic viral biology and providing a foundation for therapeutic advances. The dearth of targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), an ongoing global health crisis, underscores the importance of gaining a deeper understanding of the interactions of proteins encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we describe the use of a convenient bacterial cell-based two-hybrid (B2H) system to analyze the SARS-CoV-2 proteome. We identified 16 distinct intraviral protein-protein interactions (PPIs), involving 16 proteins. We found that many of the identified proteins interact with more than one partner. Further, our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified B2H system that permits the detection of disulfide bond-dependent PPIs in the normally reducing Escherichia coli cytoplasm, and we used this system to detect the interaction of the SARS-CoV-2 spike protein receptor-binding domain (RBD) with its cognate cell surface receptor ACE2. We then examined how the RBD-ACE2 interaction is perturbed by several RBD amino acid substitutions found in currently circulating SARS-CoV-2 variants. Our findings illustrate the utility of a genetically tractable bacterial system for probing the interactions of viral proteins and investigating the effects of emerging mutations. In principle, the system could also facilitate the identification of potential therapeutics that disrupt specific interactions of virally encoded proteins. More generally, our findings establish the feasibility of using a B2H system to detect and dissect disulfide bond-dependent interactions of eukaryotic proteins. IMPORTANCE Understanding how virally encoded proteins interact with one another is essential in elucidating basic viral biology, providing a foundation for therapeutic discovery. Here, we describe the use of a versatile bacterial cell-based system to investigate the interactions of the protein set encoded by SARS-CoV-2, the virus responsible for the current COVID-19 pandemic. We identified 16 distinct intraviral protein-protein interactions, involving 16 proteins, many of which interact with more than one partner. Our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified version of our bacterial cell-based system that permits detection of the interaction between the SARS-CoV-2 spike protein (specifically, its receptor-binding domain) and its cognate human cell surface receptor ACE2, and we investigated the effects of spike mutations found in currently circulating SARS-CoV-2 variants. Our findings illustrate the general utility of our system for probing the interactions of virally encoded proteins.

SsaV Interacts with SsaL to Control the Translocon-to-Effector Switch in the Salmonella SPI-2 Type Three Secretion System.

Nonflagellar type III secretion systems (nf T3SSs) form a cell surface needle-like structure and an associated translocon that deliver bacterial effector proteins into eukaryotic host cells. This involves a tightly regulated hierarchy of protein secretion. A switch involving SctP and SctU stops secretion of the needle protein. The gatekeeper protein SctW is required for secretion of translocon proteins and controls a second switch to start effector secretion. Salmonella enterica serovar Typhimurium encodes two T3SSs in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. The acidic vacuole containing intracellular bacteria stimulates assembly of the SPI-2 T3SS and its translocon. Sensing the nearly neutral host cytosolic pH is required for effector translocation. Here, we investigated the involvement of SPI-2-encoded proteins SsaP (SctP), SsaU (SctU), SsaV (SctV), and SsaL (SctW) in regulation of secretion. We found that SsaP and SsaU are involved in the first but not the second secretion switch. A random-mutagenesis screen identified amino acids of SsaV that regulate translocon and effector secretion. Single substitutions in subdomain 4 of SsaV or InvA (SPI-1-encoded SctV) phenocopied mutations of their corresponding gatekeepers with respect to translocon and effector protein secretion and host cell interactions. SsaL interacted with SsaV in bacteria exposed to low ambient pH but not after the pH was raised to 7.2. We propose that SsaP and SsaU enable the apparatus to become competent for a secretion switch and facilitate the SsaL-SsaV interaction. This mediates secretion of translocon proteins until neutral pH is sensed, which causes their dissociation, resulting in arrest of translocon secretion and derepression of effector translocation.IMPORTANCESalmonella Typhimurium is an intracellular pathogen that uses the SPI-2 type III secretion system to deliver virulence proteins across the vacuole membrane surrounding intracellular bacteria. This involves a tightly regulated hierarchy of protein secretion controlled by two molecular switches. We found that SPI-2-encoded proteins SsaP and SsaU are involved in the first but not the second secretion switch. We identify key amino acids of the inner membrane protein SsaV that are required to interact with the so-called gatekeeper protein SsaL and show that the dissociation of SsaV-SsaL causes the second switch, leading to delivery of effector proteins. Our results provide insights into the molecular events controlling virulence-associated type III secretion and suggest a broader model describing how the process is regulated.

Cell Density-Dependent Cytological Stage Profile and Its Application for a Screen of Cytostatic Agents Active Toward Leukemic Stem Cells.

Proliferation and expansion of leukemia is driven by leukemic stem cells (LSCs). Multidrug resistance (MDR) of LSCs is one of the main reasons of failure and relapses in acute myeloid leukemia (AML) treatment. In this study, we show that maintaining HL-60 at low cell culture density or applying a 240-day treatment with anthrapyridazone (BS-121) increased the percentage of primitive cells, which include LSCs determining the overall stage profile. This change manifested in morphology, expression of both cell surface markers and redox-state proteins, as well as mitochondrial potential. Moreover, four sublines were generated, each with unique and characteristic stage profile and cytostatic sensitivity. Cell density-induced culture alterations (affecting stage profiles) were exploited in a screen of anthrapyridazones. Among the compound tested, C-123 was the most potent against primitive cell stages while generating relatively low amounts of reactive oxygen species (ROS). Furthermore, it had low toxicity in vivo and weakly affected blood morphology of healthy mice. The cell density-dependent stage profiles could be utilized in preliminary drug screens for activity against LSCs or in construction of patient-specific platforms to find drugs effective in case of AML relapse (drug extrapolation). The correlation between ROS generation in differentiated cells and toxic effect observed in HL-60 has a potential application in myelotoxicity predictions. The discovered properties of C-123 indicate its potential application in AML treatment, specifically in conditioned myeloablation preceding allogeneic transplantation and/or ex vivo treatment preceding autologous transplantation.

Activity of acetyltransferase toxins involved in Salmonella persister formation during macrophage infection.

Non-typhoidal Salmonella strains are responsible for invasive infections associated with high mortality and recurrence in sub-Saharan Africa, and there is strong evidence for clonal relapse following antibiotic treatment. Persisters are non-growing bacteria that are thought to be responsible for the recalcitrance of many infections to antibiotics. Toxin-antitoxin systems are stress-responsive elements that are important for Salmonella persister formation, specifically during infection. Here, we report the analysis of persister formation of clinical invasive strains of Salmonella Typhimurium and Enteritidis in human primary macrophages. We show that all the invasive clinical isolates of both serovars that we tested produce high levels of persisters following internalization by human macrophages. Our genome comparison reveals that S. Enteritidis and S. Typhimurium strains contain three acetyltransferase toxins that we characterize structurally and functionally. We show that all induce the persister state by inhibiting translation through acetylation of aminoacyl-tRNAs. However, they differ in their potency and target partially different subsets of aminoacyl-tRNAs, potentially accounting for their non-redundant effect.

Molecular basis for the DNA damage induction and anticancer activity of asymmetrically substituted anthrapyridazone PDZ-7.

Anthrapyridazones, imino analogues of anthraquinone, constitute a family of compounds with remarkable anti-cancer activity. To date, over 20 derivatives were studied, of which most displayed nanomolar cytotoxicity towards broad spectrum of cancer cells, including breast, prostate and leukemic ones. BS-154, the most potent derivative, had IC50 values close to 1 nM, however, it was toxic in animal studies. Here, we characterize another anthrapyridazone, PDZ-7, which retains high cytotoxicity while being well tolerated in mice. PDZ-7 is also active in vivo against anthracycline-resistant tumor in a mouse xenograft model and induces DNA damage in proliferating cells, preferentially targeting cells in S and G2 phases of the cell cycle. Activation of Mre11-Rad50-Nbs1 (MRN) complex and phosphorylation of H2AX suggest double-stranded DNA breaks as a major consequence of PDZ-7 treatment. Consistent with this, PDZ-7 treatment blocked DNA synthesis and resulted in cell cycle arrest in late S and G2 phases. Analysis of topoisomerase IIα activity and isolation of the stabilized covalent topoisomerase IIα - DNA complex in the presence of PDZ-7 suggests that this compound is a topoisomerase IIα poison. Moreover, PDZ-7 interfered with actin polymerization, thereby implying its action as a dual inhibitor of processes critical for dividing cells. Using nuclear magnetic resonance (NMR) spectroscopy we show that PDZ-7 interacts with DNA double helix and quadruplex DNA structure. Taken together, our results suggest that PDZ-7 is a unique compound targeting actin cytoskeleton and DNA.

Atomic resolution insights into curli fiber biogenesis.

Bacteria produce functional amyloid fibers called curli in a controlled, noncytotoxic manner. These extracellular fimbriae enable biofilm formation and promote pathogenicity. Understanding curli biogenesis is important for appreciating microbial lifestyles and will offer clues as to how disease-associated human amyloid formation might be ameliorated. Proteins encoded by the curli specific genes (csgA-G) are required for curli production. We have determined the structure of CsgC and derived the first structural model of the outer-membrane subunit translocator CsgG. Unexpectedly, CsgC is related to the N-terminal domain of DsbD, both in structure and oxido-reductase capability. Furthermore, we show that CsgG belongs to the nascent class of helical outer-membrane macromolecular exporters. A cysteine in a CsgG transmembrane helix is a potential target of CsgC, and mutation of this residue influences curli assembly. Our study provides the first high-resolution structural insights into curli biogenesis.