Kadie Holsinger

mBio. 2023 Feb 13:e0316822. doi: 10.1128/mbio.03168-22. Online ahead of print.

ABSTRACT

Bacteria can adapt to stressful conditions through mutations affecting the RNA polymerase core subunits that lead to beneficial changes in transcription. In response to selection with rifampicin (RIF), mutations arise in the RIF resistance-determining region (RRDR) of rpoB that reduce antibiotic binding. These changes can also alter transcription and thereby have pleiotropic effects on bacterial fitness. Here, we studied the evolution of resistance in Bacillus subtilis to the synergistic combination of RIF and the β-lactam cefuroxime (CEF). Two independent evolution experiments led to the recovery of a single rpoB allele (S487L) that was able to confer resistance to RIF and CEF through a single mutation. Two other common RRDR mutations made the cells 32 times more sensitive to CEF (H482Y) or led to only modest CEF resistance (Q469R). The diverse effects of these three mutations on CEF resistance are correlated with differences in the expression of peptidoglycan (PG) synthesis genes and in the levels of two metabolites crucial in regulating PG synthesis, glucosamine-6-phosphate (GlcN-6-P) and UDP-N-acetylglucosamine (UDP-GlcNAc). We conclude that RRDR mutations can have widely varying effects on pathways important for cell wall biosynthesis, and this may restrict the spectrum of mutations that arise during combination therapy. IMPORTANCE Rifampicin (RIF) is one of the most valued drugs in the treatment of tuberculosis. TB treatment relies on a combination therapy and for multidrug-resistant strains may include β-lactams. Mutations in rpoB present a common route for emergence of resistance to RIF. In this study, using B. subtilis as a model, we evaluate the emergence of resistance for the synergistic combination of RIF and the β-lactam cefuroxime (CEF). One clinically relevant rpoB mutation conferred resistance to both RIF and CEF, whereas one other increased CEF sensitivity. We were able to link these CEF sensitivity phenotypes to accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc), which feedback regulates GlmS activity and thereby peptidoglycan synthesis. Further, we found that higher CEF concentrations precluded the emergence of high RIF resistance. Collectively, these results suggest that multidrug treatment regimens may limit the available pathways for the evolution of antibiotic resistance.

PMID:36779708 | DOI:10.1128/mbio.03168-22

Amino Acids. 2023 Feb 12. doi: 10.1007/s00726-023-03245-w. Online ahead of print.

ABSTRACT

The global increase in antimicrobial drug resistance has dramatically reduced the effectiveness of traditional antibiotics. Structurally diverse antibiotics are urgently needed to combat multiple-resistant bacterial infections. As part of innate immunity, antimicrobial peptides have been recognized as the most promising candidates because they comprise diverse sequences and mechanisms of action and have a relatively low induction rate of resistance. However, because of their low chemical stability, susceptibility to proteases, and high hemolytic effect, their usage is subject to many restrictions. Chemical modifications such as D-amino acid substitution, cyclization, and unnatural amino acid modification have been used to improve the stability of antimicrobial peptides for decades. Among them, a side-chain covalent bridge modification, the so-called stapled peptide, has attracted much attention. The stapled side-chain bridge stabilizes the secondary structure, induces protease resistance, and increases cell penetration and biological activity. Recent progress in computer-aided drug design and artificial intelligence methods has also been used in the design of stapled antimicrobial peptides and has led to the successful discovery of many prospective peptides. This article reviews the possible structure-activity relationships of stapled antimicrobial peptides, the physicochemical properties that influence their activity (such as net charge, hydrophobicity, helicity, and dipole moment), and computer-aided methods of stapled peptide design. Antimicrobial peptides under clinical trial: Pexiganan (NCT01594762, 2012-05-07). Omiganan (NCT02576847, 2015-10-13).

PMID:36781451 | DOI:10.1007/s00726-023-03245-w

Methods Mol Biol. 2023;2628:505-533. doi: 10.1007/978-1-0716-2978-9_30.

ABSTRACT

Antigenic peptides are commonly used in serological test settings such as enzyme-linked immunosorbent assays (ELISA) to determine reactive antibodies (ABs) from serum or plasma samples. The use of synthetic peptides provides advantages like lower production effort and easier incorporation of specific chemical modifications compared to full-length antigenic proteins. Multiplexed antibody (AB) profiling methods such as microarray technologies enable the simultaneous identification of multiple novel biomarkers for the use in early disease diagnostics, vaccine development, or monitoring of immune responses. Despite various benefits they still show major limitations which can be overcome with bead-based assay technologies like the multi-analyte profiling (xMAP) technology developed by Luminex. In this chapter we introduce our established workflow for AB profiling with a multiplexed bead-based peptide immunoassay. The workflow is based on copper-catalyzed click chemistry to immobilize designed synthetic peptides onto uniquely color-coded paramagnetic beads in an orientation-specific manner. The individual peptide-coupled beads can be distinguished by their unique emission spectra during readout in the xMAP instrument and therefore allow testing of up to 500 different antigenic peptides in one multiplexed reaction. The multistep process described in this chapter is divided into separate sections for peptide design, coupling of functionalized peptides to MagPlex beads via click chemistry, confirmation of successful peptide immobilization, processing of serum or plasma samples, or preferably purified IgG thereof, with the multiplexed bead-based peptide immunoassay and subsequent data export and analysis.

PMID:36781804 | DOI:10.1007/978-1-0716-2978-9_30

Nanoscale. 2023 Feb 13. doi: 10.1039/d2nr05421c. Online ahead of print.

ABSTRACT

The increasing emergence and dissemination of antibiotic resistance pose a severe threat to overwhelming healthcare practices worldwide. The lack of new antibacterial drugs urgently calls for alternative therapeutic strategies to combat multidrug-resistant (MDR) bacterial pathogens, especially those that survive and replicate in host cells, causing relapse and recurrence of infections. Intracellular drug delivery is a direct efficient strategy to combat invasive pathogens by increasing the accumulation of antibiotics. However, the increased accumulation of antibiotics in the infected host cells does not mean high efficacy. The difficulty of treatment lies in the efficient intracellular delivery of antibiotics to the pathogen-containing compartments. Here, we first briefly review the survival mechanisms of intracellular bacteria to facilitate the exploration of potential antibacterial targets for precise delivery. Furthermore, we provide an overview of endocytosis-mediated drug delivery systems, including the biomedical and physicochemical properties modulating the endocytosis and intracellular redistribution of antibiotics. Lastly, we summarize the targets and payloads of recently described intracellular delivery systems and their modes of action against diverse pathogenic bacteria-associated infections. This overview of endocytosis-mediated redistribution of antibiotics sheds light on the development of novel delivery platforms and alternative strategies to combat intracellular bacterial pathogens.

PMID:36779877 | DOI:10.1039/d2nr05421c

bioRxiv. 2023 Feb 4:2023.02.03.527047. doi: 10.1101/2023.02.03.527047. Preprint.

ABSTRACT

The cell envelope of Gram-negative bacteria consists of three distinct layers: the cytoplasmic membrane, a cell wall made of peptidoglycan (PG), and an asymmetric outer membrane (OM) composed of phospholipid in the inner leaflet and lipopolysaccharide (LPS) glycolipid in the outer leaflet. The PG layer has long been thought to be the major structural component of the envelope protecting cells from osmotic lysis and providing them with their characteristic shape. In recent years, the OM has also been shown to be a load-bearing layer of the cell surface that fortifies cells against internal turgor pressure. However, whether the OM also plays a role in morphogenesis has remained unclear. Here, we report that changes in LPS synthesis or modification predicted to strengthen the OM can suppress the growth and shape defects of Escherichia coli mutants with reduced activity in a conserved PG synthesis machine called the Rod system (elongasome) that is responsible for cell elongation and shape determination. Evidence is presented that OM fortification in the shape mutants restores the ability of MreB cytoskeletal filaments to properly orient the synthesis of new cell wall material by the Rod system. Our results are therefore consistent with a role for the OM in the propagation of rod shape during growth in addition to its well-known function as a diffusion barrier promoting the intrinsic antibiotic resistance of Gram-negative bacteria.

SIGNIFICANCE: The cell wall has traditionally been thought to be the main structural determinant of the bacterial cell envelope that resists internal turgor and determines cell shape. However, the outer membrane (OM) has recently been shown to contribute to the mechanical strength of Gram-negative bacterial envelopes. Here, we demonstrate that changes to OM composition predicted to increase its load bearing capacity rescue the growth and shape defects of Escherichia coli mutants defective in the major cell wall synthesis machinery that determines rod shape. Our results therefore reveal a previously unappreciated role for the OM in bacterial shape determination in addition to its well-known function as a diffusion barrier that protects Gram-negative bacteria from external insults like antibiotics.

PMID:36778245 | PMC:PMC9915748 | DOI:10.1101/2023.02.03.527047

Front Microbiol. 2023 Jan 25;14:1118529. doi: 10.3389/fmicb.2023.1118529. eCollection 2023.

ABSTRACT

The gastrointestinal tract of the human is inhabited by about 5 × 10¹³ bacteria (of about 1,000 species) as well as archaea, fungi, and viruses. Gut microbiota is known to influence the host organism, but the host may also affect the functioning of the microbiota. This bidirectional cooperation occurs in three main inter-organ signaling: immune, neural, and endocrine. Immune communication relies mostly on the cytokines released by the immune cells into circulation. Also, pathogen-associated or damage-associated molecular patterns (PAMPs or DAMPs) may enter circulation and affect the functioning of the internal organs and gut microbiota. Neural communication relies mostly on the direct anatomical connections made by the vagus nerve, or indirect connections via the enteric nervous system. The third pathway, endocrine communication, is the broadest one and includes the hypothalamic-pituitary-adrenal axis. This review focuses on presenting the latest data on the role of the gut microbiota in inter-organ communication with particular emphasis on the role of neurotransmitters (catecholamines, serotonin, gamma-aminobutyric acid), intestinal peptides (cholecystokinin, peptide YY, and glucagon-like peptide 1), and bacterial metabolites (short-chain fatty acids).

PMID:36760508 | PMC:PMC9907780 | DOI:10.3389/fmicb.2023.1118529

Front Microbiol. 2022 Sep 6;13:985871. doi: 10.3389/fmicb.2022.985871. eCollection 2022.

ABSTRACT

Beta-lactams have been excluded from tuberculosis therapy due to the intrinsic resistance of Mycobacterium tuberculosis (Mtb) to this antibiotic class, usually attributed to a potent beta-lactamase, BlaC, and to an unusually complex cell wall. In this pathogen, the peptidoglycan is cross-linked by penicillin-binding proteins (PBPs) and L,D-transpeptidases, the latter resistant to inhibition by most beta-lactams. However, recent studies have shown encouraging results of beta-lactam/beta-lactamase inhibitor combinations in clinical strains. Additional research on the mechanisms of action and resistance to these antibiotics and other inhibitors of peptidoglycan synthesis, such as the glycopeptides, is crucial to ascertain their place in alternative regimens against drug-resistant strains. Within this scope, we applied selective pressure to generate mutants resistant to amoxicillin, meropenem or vancomycin in Mtb H37Rv or Mycolicibacterium smegmatis (Msm) mc²-155. These were phenotypically characterized, and whole-genome sequencing was performed. Mutations in promising targets or orthologue genes were inspected in Mtb clinical strains to establish potential associations between altered susceptibility to beta-lactams and the presence of key genomic signatures. The obtained isolates had substantial increases in the minimum inhibitory concentration of the selection antibiotic, and beta-lactam cross-resistance was detected in Mtb. Mutations in L,D-transpeptidases and major PBPs, canonical targets, or BlaC were not found. The transcriptional regulator PhoP (Rv0757) emerged as a common denominator for Mtb resistance to both amoxicillin and meropenem, while Rv2864c, a lipoprotein with PBP activity, appears to be specifically involved in decreased susceptibility to the carbapenem. Nonetheless, the mutational pattern detected in meropenem-resistant mutants was different from the yielded by amoxicillin-or vancomycin-selected isolates, suggesting that distinct pathways may participate in increased resistance to peptidoglycan inhibitors, including at the level of beta-lactam subclasses. Cross-resistance between beta-lactams and antimycobacterials was mostly unnoticed, and Msm meropenem-resistant mutants from parental strains with previous resistance to isoniazid or ethambutol were isolated at a lower frequency. Although cell-associated nitrocefin hydrolysis was increased in some of the isolates, our findings suggest that traditional assumptions of Mtb resistance relying largely in beta-lactamase activity and impaired access of hydrophilic molecules through lipid-rich outer layers should be challenged. Moreover, the therapeutical potential of the identified Mtb targets should be explored.

PMID:36147841 | PMC:PMC9485614 | DOI:10.3389/fmicb.2022.985871

J Bacteriol. 2023 Jan 26;205(1):e0042422. doi: 10.1128/jb.00424-22. Epub 2022 Dec 21.

ABSTRACT

The peptidoglycan of mycobacteria has two types of direct cross-links, classical 4-3 cross-links that occur between diaminopimelate (DAP) and alanine residues, and nonclassical 3-3 cross-links that occur between DAP residues on adjacent peptides. The 3-3 cross-links are synthesized by the concerted action of d,d-carboxypeptidases and l,d-transpeptidases (Ldts). Mycobacterial genomes encode several Ldt proteins that can be classified into six classes based upon sequence identity. As a group, the Ldt enzymes are resistant to most β-lactam antibiotics but are susceptible to carbapenem antibiotics, with the exception of LdtC, a class 5 enzyme. In previous work, we showed that loss of LdtC has the greatest effect on the carbapenem susceptibility phenotype of Mycobacterium smegmatis (also known as Mycolicibacterium smegmatis) compared to other ldt deletion mutants. In this work, we show that a M. smegmatis mutant lacking the five ldt genes other than ldtC has a wild-type phenotype with the exception of increased susceptibility to rifampin. In contrast, a mutant lacking all six ldt genes has pleiotropic cell envelope defects, is temperature sensitive, and has increased susceptibility to a variety of antibiotics. These results indicate that LdtC is capable of functioning as the sole l,d-transpeptidase in M. smegmatis and suggest that it may represent a carbapenem-resistant pathway for peptidoglycan biosynthesis. IMPORTANCE Mycobacteria have several enzymes to catalyze nonclassical 3-3 linkages in the cell wall peptidoglycan. Understanding the biology of these cross-links is important for the development of antibiotic therapies to target peptidoglycan biosynthesis. Our work provides evidence that LdtC can function as the sole enzyme for 3-3 cross-link formation in M. smegmatis and suggests that LdtC may be part of a carbapenem-resistant l,d-transpeptidase pathway.

PMID:36541811 | PMC:PMC9879121 | DOI:10.1128/jb.00424-22

Nat Commun. 2022 Dec 27;13(1):7962. doi: 10.1038/s41467-022-35528-3.

ABSTRACT

The D,D-transpeptidase activity of penicillin-binding proteins (PBPs) is the well-known primary target of β-lactam antibiotics that block peptidoglycan polymerization. β-lactam-induced bacterial killing involves complex downstream responses whose causes and consequences are difficult to resolve. Here, we use the functional replacement of PBPs by a β-lactam-insensitive L,D-transpeptidase to identify genes essential to mitigate the effects of PBP inactivation by β-lactams in actively dividing bacteria. The functions of the 179 conditionally essential genes identified by this approach extend far beyond L,D-transpeptidase partners for peptidoglycan polymerization to include proteins involved in stress response and in the assembly of outer membrane polymers. The unsuspected effects of β-lactams include loss of the lipoprotein-mediated covalent bond that links the outer membrane to the peptidoglycan, destabilization of the cell envelope in spite of effective peptidoglycan cross-linking, and increased permeability of the outer membrane. The latter effect indicates that the mode of action of β-lactams involves self-promoted penetration through the outer membrane.

PMID:36575173 | PMC:PMC9794725 | DOI:10.1038/s41467-022-35528-3

J Bacteriol. 2023 Feb 9:e0042822. doi: 10.1128/jb.00428-22. Online ahead of print.

ABSTRACT

The dynamic composition of the peptidoglycan cell wall has been the subject of intense research for decades, yet how bacteria coordinate the synthesis of new peptidoglycan with the turnover and remodeling of existing peptidoglycan remains elusive. Diversity and redundancy within peptidoglycan synthases and peptidoglycan autolysins, enzymes that degrade peptidoglycan, have often made it challenging to assign physiological roles to individual enzymes and determine how those activities are regulated. For these reasons, peptidoglycan glycosidases, which cleave within the glycan strands of peptidoglycan, have proven veritable masters of misdirection over the years. Unlike many of the broadly conserved peptidoglycan synthetic complexes, diverse bacteria can employ unrelated glycosidases to achieve the same physiological outcome. Additionally, although the mechanisms of action for many individual enzymes have been characterized, apparent conserved homologs in other organisms can exhibit an entirely different biochemistry. This flexibility has been recently demonstrated in the context of three functions critical to vegetative growth: (i) release of newly synthesized peptidoglycan strands from their membrane anchors, (ii) processing of peptidoglycan turned over during cell wall expansion, and (iii) removal of peptidoglycan fragments that interfere with daughter cell separation during cell division. Finally, the regulation of glycosidase activity during these cell processes may be a cumulation of many factors, including protein-protein interactions, intrinsic substrate preferences, substrate availability, and subcellular localization. Understanding the true scope of peptidoglycan glycosidase activity will require the exploration of enzymes from diverse organisms with equally diverse growth and division strategies.

PMID:36757204 | DOI:10.1128/jb.00428-22

Biotechnol Adv. 2023 May-Jun;64:108108. doi: 10.1016/j.biotechadv.2023.108108. Epub 2023 Feb 3.

ABSTRACT

The engineering of potent prophylactic and therapeutic complexes has always required careful protein modification techniques with seamless capabilities. In this light, methods that favor unobstructed multivalent targeting and correct antigen presentations remain essential and very demanding. Sortase A (SrtA) transpeptidation has exhibited these attributes in various settings over the years. However, its applications for engineering avidity-inspired therapeutics and potent vaccines have yet to be significantly noticed, especially in this era where active targeting and multivalent nanomedications are in great demand. This review briefly presents the SrtA enzyme and its associated transpeptidation activity and describes interesting sortase-mediated protein engineering and chemistry approaches for achieving multivalent therapeutic and antigenic responses. The review further highlights advanced applications in targeted delivery systems, multivalent therapeutics, adoptive cellular therapy, and vaccine engineering. These innovations show the potential of sortase-mediated techniques in facilitating the development of simple plug-and-play nanomedicine technologies against recalcitrant diseases and pandemics such as cancer and viral infections.

PMID:36740026 | DOI:10.1016/j.biotechadv.2023.108108

J Med Chem. 2023 Feb 5. doi: 10.1021/acs.jmedchem.2c01344. Online ahead of print.

ABSTRACT

The current global issue of antibiotic resistance is serious, and there is an urgent requirement of developing novel antibiotics. Octapeptins have recently regained interest because of their activities against resistant Gram-negative bacteria. We synthesized four natural octapeptins and 33 derivatives with diverse polarity, amphiphilicity, and acid-base properties by solid-phase synthesis and investigated their in vitro antibacterial activity and renal cytotoxicity. We also assessed the structure-activity relationship and structure-toxicity relationship of the cyclic lipopeptide compounds. Some compounds showed increased activity against Gram-negative and/or Gram-positive bacteria, with improved renal cytotoxicity. C-02 showed remarkable in vitro antibacterial activity and low renal cytotoxicity. We found that C-02 showed high antibacterial activity against Escherichia coli in vivo and manifested its effects preliminarily by increasing outer membrane permeability. Therefore, C-02 might be a new antibiotic lead compound with not only high efficacy but also low renal cytotoxicity.

PMID:36739537 | DOI:10.1021/acs.jmedchem.2c01344

Biochem Biophys Res Commun. 2023 Jan 30;648:66-71. doi: 10.1016/j.bbrc.2023.01.094. Online ahead of print.

ABSTRACT

Antimicrobial peptides (AMPs) are vital components of the nonspecific immune system that represent a promising broad-spectrum alternative to conventional antibiotics. Several short cationic antimicrobial peptides show highly effective antibacterial activity and low hemolytic activity, which are based on the action of a few critical amino acids, such as phenylalanine (F) and lysine (K). Previous studies have reported that Fmoc-based phenylalanine peptides possess appreciable antibacterial potency against Gram-positive bacteria, but their ability to kill Gram-negative bacteria was suboptimal. In this study, we designed and prepared a series of Fmoc-K_nF peptide (n = 1-3) series by adding lysine motifs to strengthen their broad-spectrum antibacterial activity. The effect was investigated that the amount of lysine in Fmoc-F peptides on their antibacterial properties and hemolytic activities. Our results showed that the Fmoc-KKF peptide holds the strongest antimicrobial activity against both Gram-positive and negative bacteria among all designed peptides, as well as low hemolytic activity. These results provide support for the general strategy of enhancing the broad-spectrum antibacterial activity of AMPs through increased lysine content.

PMID:36736093 | DOI:10.1016/j.bbrc.2023.01.094

J Med Chem. 2023 Feb 5. doi: 10.1021/acs.jmedchem.2c00757. Online ahead of print.

ABSTRACT

Clinically, antibiotics are widely used to treat infectious diseases; however, excessive drug abuse and overuse exacerbate the prevalence of drug-resistant bacterial pathogens, making the development of novel antibiotics extremely difficult. Antimicrobial peptide (AMP) is one of the most promising candidates for overcoming bacterial resistance owing to its unique structure and mechanism of action. This study examines the development of small molecular mimetics of AMPs over the past two decades. These mimetics can selectively disrupt membranes, which are the characteristic antibacterial mechanism of AMPs. In addition, the advantages and disadvantages of small AMP mimetics are discussed. The small molecular mimetics of AMPs are anticipated to garner interest and investment in discovering new antibiotics. This Perspective will assist in revitalizing the golden age of antibiotics in the current era of combating bacterial resistance.

PMID:36739538 | DOI:10.1021/acs.jmedchem.2c00757

Front Immunol. 2023 Jan 19;14:1088501. doi: 10.3389/fimmu.2023.1088501. eCollection 2023.

ABSTRACT

Staphylococcus aureus infection is a severe public health concern with the growing number of multidrug-resistant strains. S. aureus can circumvent the defense mechanisms of host immunity with the aid of multiple virulence factors. An efficacious multicomponent vaccine targeting diverse immune evasion strategies developed by S. aureus is thus crucial for its infection control. In this study, we exploited the SpyCatcher-SpyTag system to engineer bacterial outer membrane vesicles (OMVs) for the development of a multitargeting S. aureus click vaccine. We decorated OMVs with surface exposed SpyCatcher via a truncated OmpA(a.a 1-155)-SpyCatcher fusion. The engineered OMVs can flexibly bind with various SpyTag-fused S. aureus antigens to generate an OMV-based click vaccine. Compared with antigens mixed with alum adjuvant, the click vaccine simultaneously induced more potent antigen-specific humoral and Th1-based cellular immune response, which afforded protection against S. aureus Newman lethal challenge in a mouse model. Our study provided a flexible and versatile click vaccine strategy with the potential for fighting against emerging S. aureus clinical isolates.

PMID:36742310 | PMC:PMC9892643 | DOI:10.3389/fimmu.2023.1088501

Front Cell Infect Microbiol. 2023 Jan 6;12:1085946. doi: 10.3389/fcimb.2022.1085946. eCollection 2022.

ABSTRACT

Combination therapy is necessary to treat tuberculosis to decrease the rate of disease relapse and prevent the acquisition of drug resistance, and shorter regimens are urgently needed. The adaptation of Mycobacterium tuberculosis to various lesion microenvironments in infection induces various states of slow replication and non-replication and subsequent antibiotic tolerance. This non-heritable tolerance to treatment necessitates lengthy combination therapy. Therefore, it is critical to develop combination therapies that specifically target the different types of drug-tolerant cells in infection. As new tools to study drug combinations earlier in the drug development pipeline are being actively developed, we must consider how to best model the drug-tolerant cells to use these tools to design the best antibiotic combinations that target those cells and shorten tuberculosis therapy. In this review, we discuss the factors underlying types of drug tolerance, how combination therapy targets these populations of bacteria, and how drug tolerance is currently modeled for the development of tuberculosis multidrug therapy. We highlight areas for future studies to develop new tools that better model drug tolerance in tuberculosis infection specifically for combination therapy testing to bring the best drug regimens forward to the clinic.

PMID:36733851 | PMC:PMC9888313 | DOI:10.3389/fcimb.2022.1085946

Infect Chemother. 2023 Jan 6. doi: 10.3947/ic.2022.0145. Online ahead of print.

ABSTRACT

Outer membrane vesicles (OMVs) are spherical bilayered nanoparticles derived from the outer layer of Gram-negative bacteria. Bacteria communicate with nearby bacteria, their environment, and the cells of their host by secreting OMVs, which are essential for their survival. OMVs also play a critical role in bacterial pathogenesis since they are loaded with virulence factors, toxins, and enzymes. OMVs may modulate the immune response of the host by initiating inflammation through cytokine production and activating the innate immune response. OMVs also contribute to the resistance of bacteria to antibiotics by carrying antibiotic-degrading enzymes and acting as natural protection barriers. Concerns have also been raised regarding OMVs mediating the transfer of antibiotic resistance. Due to their advantageous properties, OMVs are attractive platforms for vaccine discovery and drug delivery research. In this review, we discuss the fundamental structure and biogenesis mechanisms of OMVs as well as their multifaceted roles in bacterial infection pathogenesis and host immune responses. We also discuss application examples of OMVs.

PMID:36731499 | DOI:10.3947/ic.2022.0145

mBio. 2023 Feb 1:e0355822. doi: 10.1128/mbio.03558-22. Online ahead of print.

ABSTRACT

Almost all bactericidal drugs require bacterial replication and/or metabolic activity for their killing activity. When these processes are inhibited by bacteriostatic antibiotics, bacterial killing is significantly reduced. One notable exception is the lipopeptide antibiotic daptomycin, which has been reported to efficiently kill growth-arrested bacteria. However, these studies employed only short periods of growth arrest (<1 h), which may not fully represent the duration of growth arrest that can occur in vivo. We found that a growth inhibitory concentration of the protein synthesis inhibitor tetracycline led to a time-dependent induction of daptomycin tolerance in S. aureus, with an approximately 100,000-fold increase in survival after 16 h of growth arrest, relative to exponential-phase bacteria. Daptomycin tolerance required glucose and was associated with increased production of the cell wall polymers peptidoglycan and wall-teichoic acids. However, while the accumulation of peptidoglycan was required for daptomycin tolerance, only a low abundance of wall teichoic acid was necessary. Therefore, whereas tolerance to most antibiotics occurs passively due to a lack of metabolic activity and/or replication, daptomycin tolerance arises via active cell wall remodelling. IMPORTANCE Understanding why antibiotics sometimes fail to cure infections is fundamental to improving treatment outcomes. This is a major challenge when it comes to Staphylococcus aureus because this pathogen causes several different chronic or recurrent infections. Previous work has shown that a lack of replication, as often occurs during infection, makes bacteria tolerant of most bactericidal antibiotics. However, one antibiotic that has been reported to kill nonreplicating bacteria is daptomycin. In this work, we show that the growth arrest of S. aureus does in fact lead to daptomycin tolerance, but it requires time, nutrients, and biosynthetic pathways, making it distinct from other types of antibiotic tolerance that occur in nonreplicating bacteria.

PMID:36722949 | DOI:10.1128/mbio.03558-22

Nat Microbiol. 2023 Jan 30. doi: 10.1038/s41564-022-01317-3. Online ahead of print.

ABSTRACT

At the end of a lytic bacteriophage replication cycle in Gram-positive bacteria, peptidoglycan-degrading endolysins that cause explosive cell lysis of the host can also attack non-infected bystander cells. Here we show that in osmotically stabilized environments, Listeria monocytogenes can evade phage predation by transient conversion to a cell wall-deficient L-form state. This L-form escape is triggered by endolysins disintegrating the cell wall from without, leading to turgor-driven extrusion of wall-deficient, yet viable L-form cells. Remarkably, in the absence of phage predation, we show that L-forms can quickly revert to the walled state. These findings suggest that L-form conversion represents a population-level persistence mechanism to evade complete eradication by phage attack. Importantly, we also demonstrate phage-mediated L-form switching of the urinary tract pathogen Enterococcus faecalis in human urine, which underscores that this escape route may be widespread and has important implications for phage- and endolysin-based therapeutic interventions.

PMID:36717719 | DOI:10.1038/s41564-022-01317-3

ACS Infect Dis. 2023 Jan 30. doi: 10.1021/acsinfecdis.2c00622. Online ahead of print.

ABSTRACT

The rise of antibiotic resistance among skin-infecting pathogens poses an urgent threat to public health and has fueled the search for new therapies. Enhancing the potency of currently used antibiotics is an alternative for the treatment of infections caused by drug-resistant pathogens. In this study, we aimed to identify a small molecule that can potentiate currently used antibiotics. IITR00693 (2-aminoperimidine), a novel antibacterial small molecule, potentiates the antibacterial activity of polymyxin B against Staphylococcus aureus and Pseudomonas aeruginosa. Herein, we investigated in detail the mode of action of this interaction and the molecule's capability to combat soft-tissue infections caused by S. aureus and P. aeruginosa. A microdilution checkerboard assay was performed to determine the synergistic interaction between polymyxin B and IITR00693 in clinical isolates of S. aureus and P. aeruginosa. Time-kill kinetics, post-antibiotic effect, and resistance generation studies were performed to assess the pharmacodynamics of the combination. Assays based on different fluorescent probes were performed to decipher the mechanism of action of this combination. The in vivo efficacy of the IITR00693-polymyxin B combination was determined in a murine acute wound infection model. IITR00693 exhibited broad-spectrum antibacterial activity. IITR00693 potentiated polymyxin B and colistin against polymyxin-resistant S. aureus. IITR00693 prevented the generation of resistant mutants against multiple antibiotics. The IITR00693-polymyxin B combination decreased the S. aureus count by >3 log₁₀ CFU in a murine acute wound infection model. IITR00693 is a potential and promising candidate for the treatment of soft-tissue infections along with polymyxins.

PMID:36716174 | DOI:10.1021/acsinfecdis.2c00622

Microbiol Spectr. 2023 Jan 31:e0319722. doi: 10.1128/spectrum.03197-22. Online ahead of print.

ABSTRACT

The majority of preclinical research has shown that Mycobacterium tuberculosis can modify host lipids in various ways. To boost its intramacrophage survival, M. tuberculosis causes host lipids to build up, resulting in the development of lipid-laden foam cells. M. tuberculosis binds to and enters the macrophage via the cell membrane cholesterol. Aggregation of cholesterol in the cell wall of M. tuberculosis and an increase in vascularity at the granuloma site reduce the permeability of rifampicin and isoniazid concentrations. However, very few studies have assessed the effect of statins on drug penetration. Here, we used atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, to observe its effect on the bacterial burden by increasing the drug concentration at the infection site. We looked into how atorvastatin could be used in conjunction with first-line drugs to promote drug permeation. In this study, we detected an accumulation of drugs at the peripheral sites of the lungs and impaired drug distribution to the diseased sites. The efficacy of antituberculosis drugs, with atorvastatin as an adjunct, on the viability of M. tuberculosis cells was demonstrated. A nontoxic statin dosage established phenotypic and normal granuloma vasculature and showed an additive effect with rifampicin and isoniazid. Our data show that statins help to reduce the tuberculosis bacterial burden. Our findings reveal that the bacterial load is connected with impaired drug permeability resulting from lipid accumulation in the bacterial cell wall. Statin therapy combined with antituberculosis medications have the potential to improve treatment in tuberculosis patients. IMPORTANCE Mycobacterium tuberculosis binds to and enters the macrophage via the cell membrane cholesterol. M. tuberculosis limits phagosomal maturation and activation without engaging in phagocytosis. Aggregation of cholesterol in the cell wall of M. tuberculosis and an increase in the vascularity at the granuloma site reduce the permeability of rifampicin and isoniazid concentrations. However, very few studies have assessed the effect of statins on drug penetration, which can be increased through a reduction in cholesterol and vascularity. Herein, we used atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, to observe its effect on bacterial burden through increasing the drug concentration at the infection site. Our main research goal is to diminish mycobacterial dissemination and attenuate bacterial growth by increasing drug permeability.

PMID:36719189 | DOI:10.1128/spectrum.03197-22