Brianna Dalesandro

Publication date: Available online 4 September 2020

Source: Cell

Author(s): Pablo F. Cañete, Carola G. Vinuesa

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PhotoPROTACs: A Novel Biotechnology for Cancer Treatment.

Trends Cell Biol. 2020 Aug 24;:

Authors: Wang ZW, Liu Y, Zhu X

Abstract
PROteolysis-TArgeting Chimeras (PROTACs) have been developed for targeting specific protein destruction. Two recent studies in Science Advances by Liu et al. and Reynders et al. reported a novel technology, PHOtochemically TArgeting Chimeras (PHOTACs) or opto-PROTAC, which is light-induced control of protein degradation. This new approach might lead to precision therapeutics in patients with cancer.

PMID: 32855019 [PubMed - as supplied by publisher]

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d-Serine and d-Alanine Regulate Adaptive Foraging Behavior in Caenorhabditis elegans via the NMDA Receptor.

J Neurosci. 2020 Aug 27;:

Authors: Saitoh Y, Katane M, Miyamoto T, Sekine M, Sakai-Kato K, Homma H

Abstract
d-Serine is a coagonist for NMDA-type glutamate receptors and is thus important for higher brain function. d-Serine is synthesized by serine racemase and degraded by d-amino acid oxidase. However, the significance of these enzymes and the relevant functions of d-amino acids remain unclear. Here, we show that in the nematode Caenorhabditis elegans, the serine racemase homolog SERR-1 and d-amino acid oxidase DAAO-1 control an adaptive foraging behavior. Similar to many organisms, C. elegans immediately initiates local search for food when transferred to a new environment. With prolonged food deprivation, the worms exhibit a long-range dispersal behavior as the adaptive foraging strategy. We found that serr-1 deletion mutants did not display this behavior, whereas daao-1 deletion mutants immediately engaged in long-range dispersal after food removal. A quantitative analysis of d-amino acids indicated that d-serine and d-alanine are both synthesized and suppressed during food deprivation. A behavioral pharmacological analysis showed that the long-range dispersal behavior requires NMDA receptor desensitization. Long-term pretreatment with d-alanine, as well as with an NMDA receptor agonist, expanded the area searched by wild-type worms immediately after food removal, whereas pretreatment with d-serine did not. We propose that d-serine and d-alanine are endogenous regulators that cooperatively induce the long-range dispersal behavior in C. elegans through actions on the NMDA receptor.SIGNIFICANCE STATEMENTIn mammals, d-serine functions as an important neuromodulator of the NMDA-type glutamate receptor, which regulates higher brain functions. In Caenorhabditis elegans, previous studies failed to clearly define the physiological significance of d-serine, d-alanine and their metabolic enzymes. In this study, we found that these d-amino acids and their associated enzymes are active during food deprivation, leading to an adaptive foraging behavior. We also found that this behavior involved NMDA receptor desensitization.

PMID: 32855271 [PubMed - as supplied by publisher]

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Click-to-Capture: A method for enriching viable Staphylococcus aureus using bio-orthogonal labeling of surface proteins.

PLoS One. 2020;15(6):e0234542

Authors: Shalizi A, Wiegers TN, Maamar H

Abstract
Staphylococcus aureus is one of the principal causative agents of bacteremia which can progress to sepsis. Rapid diagnostic tests for identification and antibiotic resistance profiling of S. aureus would improve patient outcomes and antibiotic stewardship, but existing methods require a lengthy culture step to obtain enough material for testing. Complexity of the host matrix, where pathogenic microbes are often present, also interferes with many diagnostic methods. Here, we describe a straightforward and rapid method for enriching viable S. aureus using bio-orthogonal, or "click," chemistry methods. Bacteria labeled in this manner can potentially be cultured, interrogated using molecular methods for pathogen identification, or used to test antibiotic susceptibility.

PMID: 32555702 [PubMed - indexed for MEDLINE]

Antibodies may bind to bacterial pathogens or their toxins to control infections, and their effector activity is mediated through the recruitment of complement component C1q or the engagement with Fcγ receptors (FcγRs). For bacterial pathogens that rely on a single toxin to cause disease, immunity correlates with toxin neutralization. Most...

Localization and tracking of individual membrane receptors on living cells was achieved upon transient binding of aptamer probes through minimally invasive solution diffusion. Probe affinity was modulated by rational engineering of the aptamer sequence. Detection of single molecules is linked to binding affinity, so probes with different affinity provide readouts on different properties, such as diffusive dynamics and receptor density levels.

Abstract

Tumor cell‐surface markers are usually overexpressed or mutated protein receptors for which spatiotemporal regulation differs between and within cancers. Single‐molecule fluorescence imaging can profile individual markers in different cellular contexts with molecular precision. However, standard single‐molecule imaging methods based on overexpressed genetically encoded tags or cumbersome probes can significantly alter the native state of receptors. We introduce a live‐cell points accumulation for imaging in nanoscale topography (PAINT) method that exploits aptamers as minimally invasive affinity probes. Localization and tracking of individual receptors are based on stochastic and transient binding between aptamers and their targets. We demonstrated single‐molecule imaging of a model tumor marker (EGFR) on a panel of living cancer cells. Affinity to EGFR was finely tuned by rational engineering of aptamer sequences to define receptor motion and/or native receptor density.

Nature Chemical Biology, Published online: 17 August 2020; doi:10.1038/s41589-020-0622-x

An αHER2 antibody–neuraminidase conjugate, which selectively targets the removal of sialic acids from glycans on breast cancer cells, bypasses a glycoimmune checkpoint and enhances tumor cell killing by the host immune system.

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Cancer-eating immune cells kitted out with CARs.

Nat Biotechnol. 2020 05;38(5):509-511

Authors: Dolgin E

PMID: 32393919 [PubMed - indexed for MEDLINE]

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Unexpected cell wall alteration-mediated bactericidal activity of the antifungal caspofungin against vancomycin-resistant Enterococcus faecium.

Antimicrob Agents Chemother. 2020 Aug 10;:

Authors: Isnard C, Hernandez SB, Guérin F, Joalland F, Goux D, Gravey F, Auzou M, Enot D, Meignen P, Giard JC, Cava F, Cattoir V

Abstract
Enterococcus faecium has become a major opportunistic pathogen with the emergence of vancomycin-resistant enterococci (VRE). As part of the gut microbiota, they have to cope with numerous stresses including effects of antibiotics and other xenobiotics, especially in patients hospitalized in intensive care units (ICUs) who receive many medications. The aim of this study was to investigate the impact of the most prescribed xenobiotics for ICU patients on fitness, pathogenicity and antimicrobial resistance of the vanB-positive E. faecium Aus0004 reference strain. Several phenotypic analyses were carried out and we observed that caspofungin, an antifungal agent belonging to the echinocandins family, had an important effect on E. faecium growth in vitro. We confirmed this effect by electron microscopy and peptidoglycan analysis and showed that, even at a subinhibitory concentration (¼ × MIC, 8 mg/L), caspofungin had an impact on cell wall organization especially in abundance of some muropeptide precursors. By RNA-seq, it was also shown that around 20% of the transcriptome were altered in the presence of caspofungin with 321 and 259 significantly upregulated and downregulated genes, respectively. Since the fungal target of caspofungin (i.e., beta-(1,3)-glucan synthase) was absent in bacteria, the mechanistic pathway of caspofungin activity was investigated. The repression of genes involved in the pyruvate metabolism seemed to have a drastic impact on bacterial cell viability while decrease of glycerol metabolism could explain the conformational modifications of peptidoglycan. This is the first report of caspofungin antibacterial activity against E. faecium, highlighting the potential impact of non-antibiotic xenobiotics against bacterial pathogens.

PMID: 32778553 [PubMed - as supplied by publisher]

MnO₂ nanoflowers were modified on the cell surface of electrochemically active bacteria, S. oneidensis MR‐1. The biohybrids, which couple bacterial respiration with tumor metabolism, can catabolize intercellular lactate and prevent intracellular lactate production in the tumor, thereby inducing significant tumor inhibition.

Abstract

By leveraging the ability of Shewanella oneidensis MR‐1 (S. oneidensis MR‐1) to anaerobically catabolize lactate through the transfer of electrons to metal minerals for respiration, a lactate‐fueled biohybrid (Bac@MnO₂) was constructed by modifying manganese dioxide (MnO₂) nanoflowers on the S. oneidensis MR‐1 surface. The biohybrid Bac@MnO₂ uses decorated MnO₂ nanoflowers as electron receptor and the tumor metabolite lactate as electron donor to make a complete bacterial respiration pathway at the tumor sites, which results in the continuous catabolism of intercellular lactate. Additionally, decorated MnO₂ nanoflowers can also catalyze the conversion of endogenous hydrogen peroxide (H₂O₂) into generate oxygen (O₂), which could prevent lactate production by downregulating hypoxia‐inducible factor‐1α (HIF‐1α) expression. As lactate plays a critical role in tumor development, the biohybrid Bac@MnO₂ could significantly inhibit tumor progression by coupling bacteria respiration with tumor metabolism.

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Tumor-targeted pH-low insertion peptide delivery of theranostic gadolinium nanoparticles for image-guided nanoparticle-enhanced radiation therapy.

Transl Oncol. 2020 Aug 04;13(11):100839

Authors: Liu W, Deacon J, Yan H, Sun B, Liu Y, Hegan D, Li Q, Coman D, Parent M, Hyder F, Roberts K, Nath R, Tillement O, Engelman D, Glazer P

Abstract
Tumor targeting studies using metallic nanoparticles (NPs) have shown that the enhanced permeability and retention effect may not be sufficient to deliver the amount of intratumoral and intracellular NPs needed for effective in vivo radiosensitization. This work describes a pH-Low Insertion Peptide (pHLIP) targeted theranostic agent to enable image-guided NP-enhanced radiotherapy using a clinically feasible amount of injected NPs. Conventional gadolinium (Gd) NPs were conjugated to pHLIPs and evaluated in vitro for radiosensitivity and in vivo for mouse MRI. Cultured A549 human lung cancer cells were incubated with 0.5 mM of pHLIP-GdNP or conventional GdNP. Mass spectrometry showed 78-fold more cellular Gd uptake with pHLIP-GdNPs, and clonogenic survival assays showed 44% more enhanced radiosensitivity by 5 Gy irradiation with pHLIP-GdNPs at pH 6.2. In contrast to conventional GdNPs, MR imaging of tumor-bearing mice showed pHLIP-GdNPs had a long retention time in the tumor (>9 h), suitable for radiotherapy, and penetrated into the poorly-vascularized tumor core. The Gd-enhanced tumor corresponded with low-pH areas also independently measured by an in vivo molecular MRI technique. pHLIPs actively target cell surface acidity from tumor cell metabolism and deliver GdNPs into cells in solid tumors. Intracellular delivery enhances the effect of short-range radiosensitizing photoelectrons and Auger electrons. Because acidity is a general hallmark of tumor cells, the delivery is more general than antibody targeting. Imaging the in vivo NP biodistribution and more acidic (often more aggressive) tumors has the potential for quantitative radiotherapy treatment planning and pre-selecting patients who will likely benefit more from NP radiation enhancement.

PMID: 32763504 [PubMed - as supplied by publisher]

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Identification of a Novel Linear B Cell Epitope on the Sao Protein of Streptococcus suis Serotype 2.

Front Immunol. 2020;11:1492

Authors: Wang J, Dong R, Zou P, Chen Y, Li N, Wang Y, Zhang T, Pan X

Abstract
Surface antigen one (Sao) protein is a bacterial surface protein identified in the important zoonotic pathogen Streptococcus suis serotype 2 (S. suis 2) during an extensive search for functional proteins. The Sao protein is anchored to the bacterial cell wall by the LPVTG motif and is widely distributed in many S. suis serotypes. In this paper, we present the immunodominant epitope peptide of the Sao protein that is recognized by BALB/c antibodies against the Sao protein: 355SEKQMPSVVNENAVTPEKQMTNKENDNIET384 (location Sao355-384). To determine the core epitope recognized by antibodies, we prepared truncation peptide libraries. Analyses of the immunoreactivity of truncation peptides with anti-Sao355-384 serum revealed that the most immunoreactive sequence was 355SEKQMPSVVNENAVTPEK372 (location Sao355-372). Moreover, we observed that this core epitope also showed good specificity based on the ratio of reactivity with serum from S. suis-positive patients compared to serum from S. suis-negative patients. Our results point to the potential of using the Sao355-372 peptide in diagnostic assays to determine S. suis infection in humans.

PMID: 32765516 [PubMed - as supplied by publisher]

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A synthetic peptide sensitizes multi-drug resistant Pseudomonas aeruginosa to antibiotics for more than two hours and permeabilizes its envelope for twenty hours.

J Biomed Sci. 2020 Aug 06;27(1):85

Authors: Rázquin-Olazarán I, Shahrour H, Martínez-de-Tejada G

Abstract
BACKGROUND: Pseudomonas aeruginosa is a Gram-negative pathogen that frequently causes life-threatening infections in immunocompromised patients. We previously showed that subinhibitory concentrations of short synthetic peptides permeabilize P. aeruginosa and enhance the lethal action of co-administered antibiotics.
METHODS: Long-term permeabilization caused by exposure of multidrug-resistant P. aeruginosa strains to peptide P4-9 was investigated by measuring the uptake of several antibiotics and fluorescent probes and by using confocal imaging and atomic force microscopy.
RESULTS: We demonstrated that P4-9, a 13-amino acid peptide, induces a growth delay (i.e. post-antibiotic effect) of 1.3 h on a multidrug-resistant P. aeruginosa clinical isolate. Remarkably, when an independently P4-9-treated culture was allowed to grow in the absence of the peptide, cells remained sensitive to subinhibitory concentrations of antibiotics such as ceftazidime, fosfomycin and erythromycin for at least 2 h. We designated this persistent sensitization to antibiotics occurring in the absence of the sensitizing agent as Post-Antibiotic Effect associated Permeabilization (PAEP). Using atomic force microscopy, we showed that exposure to P4-9 induces profound alterations on the bacterial surface and that treated cells need at least 2 h of growth to repair those lesions. During PAEP, P. aeruginosa mutants overexpressing either the efflux pump MexAB-OprM system or the AmpC β-lactamase were rendered sensitive to antibiotics that are known substrates of those mechanisms of resistance. Finally, we showed for the first time that the descendants of bacteria surviving exposure to a membrane disturbing peptide retain a significant level of permeability to hydrophobic compounds, including propidium iodide, even after 20 h of growth in the absence of the peptide.
CONCLUSIONS: The phenomenon of long-term sensitization to antibiotics shown here may have important therapeutic implications for a combined peptide-antibiotic treatment because the peptide would not need to be present to exert its antibiotic enhancing activity as long as the target organism retains sensitization to the antibiotic.

PMID: 32762680 [PubMed - as supplied by publisher]

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Peptidoglycan biosynthesis and remodeling revisited.

Adv Appl Microbiol. 2020;112:67-103

Authors: Shaku M, Ealand C, Matlhabe O, Lala R, Kana BD

Abstract
The bacterial peptidoglycan layer forms a complex mesh-like structure that surrounds the cell, imparting rigidity to withstand cytoplasmic turgor and the ability to tolerate stress. As peptidoglycan has been the target of numerous clinically successful antimicrobials such as penicillin, the biosynthesis, remodeling and recycling of this polymer has been the subject of much interest. Herein, we review recent advances in the understanding of peptidoglycan biosynthesis and remodeling in a variety of different organisms. In order for bacterial cells to grow and divide, remodeling of cross-linked peptidoglycan is essential hence, we also summarize the activity of important peptidoglycan hydrolases and how their functions differ in various species. There is a growing body of evidence highlighting complex regulatory mechanisms for peptidoglycan metabolism including protein interactions, phosphorylation and protein degradation and we summarize key recent findings in this regard. Finally, we provide an overview of peptidoglycan recycling and how components of this pathway mediate resistance to drugs. In the face of growing antimicrobial resistance, these recent advances are expected to uncover new drug targets in peptidoglycan metabolism, which can be used to develop novel therapies.

PMID: 32762868 [PubMed - as supplied by publisher]

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FmhA and FmhC of Staphylococcus aureus incorporate serine residues into peptidoglycan crossbridges.

J Biol Chem. 2020 Aug 05;:

Authors: Willing S, Dyer E, Schneewind O, Missiakas D

Abstract
Staphylococcal peptidoglycan is characterized by pentaglycine crossbridges that are crosslinked between adjacent wall peptides by Penicillin-Binding Proteins (PBPs) to confer robustness and flexibility. In Staphylococcus aureus, pentaglycine crossbridges are synthesized by three proteins: FemX adds the first glycine and the homodimers FemA and FemB sequentially add two Gly-Gly dipeptides. Occasionally, serine residues are also incorporated into the crossbridges by enzymes that have heretofore not been identified. Here, we show that the FemA/FemB homologues FmhA and FmhC pair with FemA and FemB to incorporate Gly-Ser dipeptides into crossbridges and to confer resistance to lysostaphin, a secreted bacteriocin that cleaves the pentaglycine-crossbridge. FmhA incorporates serine residues at positions 3 and 5 of the crossbridge. In contrast, FmhC incorporates a single serine at position 5. Serine incorporation also lowers resistance toward oxacillin, an antibiotic that targets PBPs, in both methicillin-sensitive and methicillin-resistant strains of S. aureus.  FmhC is encoded by a gene immediately adjacent to lytN which specifies a hydrolase that cleaves the bond between the fifth glycine of crossbridges and the alanine of the adjacent stem peptide. In this manner, LytN facilitates the separation of daughter cells. Cell wall damage induced upon lytN overexpression can be alleviated by overexpression of fmhC.  Together, these observations suggest that FmhA and FmhC generate peptidoglycan crossbridges with unique serine patterns that provide protection from endogenous murein hydrolases governing cell division and from bacteriocins produced by microbial competitors.

PMID: 32759309 [PubMed - as supplied by publisher]

Scientists at Nanyang Technological University, Singapore (NTU Singapore) have developed a synthetic peptide that can make multidrug-resistant bacteria sensitive to antibiotics again when used together with traditional antibiotics, offering hope for the prospect of a combination treatment strategy to tackle certain antibiotic-tolerant infections.

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Targeting CD146 using folic acid-conjugated nanoparticles and suppression of tumor growth in a mouse glioma model.

J Neurosurg. 2020 Jul 24;:1-11

Authors: Fukui N, Yawata T, Nakajo T, Kawanishi Y, Higashi Y, Yamashita T, Aratake T, Honke K, Ueba T

Abstract
OBJECTIVE: Glioma stem cells (GSCs) are responsible for tumor initiation, therapeutic resistance, and recurrence. CD146 is mainly expressed in dividing GSCs and regulates cell cycle progression. However, the evaluation of the efficacy of targeted therapy against CD146 in vivo remains to be investigated. In this study, the authors aimed to develop gene therapy targeting GSCs using chitosan oligosaccharide lactate (COL) nanoparticles (NPs) conjugated with folic acid-polyethylene glycol (FA-PEG-COL NPs) for in vitro and in vivo delivery of CD146 small-interfering RNA (siCD146) and to determine the effect of CD146 knockdown on tumor growth.
METHODS: To examine the uptake of NPs by tumor cells, immunofluorescence staining, flow cytometry, and in vivo imaging were performed. The knockdown effect of siCD146 was measured by western blot and water-soluble tetrazolium salt-8 assay in mouse glioma cells. The efficacy of siRNA therapy-targeted GSCs was evaluated by monitoring tumor growth through in vivo imaging and histological analysis.
RESULTS: In vivo accumulation of the FA-PEG-COL NPs in subcutaneous and intracranial gliomas following NP administration via a mouse tail vein was observed. Additionally, in vitro delivery of siCD146 ionically cross-linked NPs, reduced CD146 levels, and suppressed growth in the glioma tumor sphere. Evaluation of the in vivo therapeutic effects of siCD146-cross-linked NPs in a mouse glioma model revealed significant suppression of intracranial tumor growth, with complete removal of the tumor observed in some mice on histological examination. Furthermore, delivery of siCD146 significantly reduced the Ki-67 index in residual tumor tissues relative to that in control mice.
CONCLUSIONS: CD146 is a potential therapeutic target, and folic acid-conjugated NPs delivering siRNA may facilitate gene therapy in malignant gliomas.

PMID: 32707539 [PubMed - as supplied by publisher]

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Modulation of Host Lipid Pathways by Pathogenic Intracellular Bacteria.

Pathogens. 2020 Jul 28;9(8):

Authors: Allen PE, Martinez JJ

Abstract
Lipids are a broad group of molecules required for cell maintenance and homeostasis. Various intracellular pathogens have developed mechanisms of modulating and sequestering host lipid processes for a large array of functions for both bacterial and host cell survival. Among the host cell lipid functions that intracellular bacteria exploit for infection are the modulation of host plasma membrane microdomains (lipid rafts) required for efficient bacterial entry; the recruitment of specific lipids for membrane integrity of intracellular vacuoles; and the utilization of host lipid droplets for the regulation of immune responses and for energy production through fatty acid β-oxidation and oxidative phosphorylation. The majority of published studies on the utilization of these host lipid pathways during infection have focused on intracellular bacterial pathogens that reside within a vacuole during infection and, thus, have vastly different requirements for host lipid metabolites when compared to those intracellular pathogens that are released into the host cytosol upon infection. Here we summarize the mechanisms by which intracellular bacteria sequester host lipid species and compare the modulation of host lipid pathways and metabolites during host cell infection by intracellular pathogens residing in either a vacuole or within the cytosol of infected mammalian cells. This review will also highlight common and unique host pathways necessary for intracellular bacterial growth that could potentially be targeted for therapeutic intervention.

PMID: 32731350 [PubMed]

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Antibodies Inhibiting the Type III Secretion System of Gram-Negative Pathogenic Bacteria.

Antibodies (Basel). 2020 Jul 27;9(3):

Authors: Hotinger JA, May AE

Abstract
Pathogenic bacteria are a global health threat, with over 2 million infections caused by Gram-negative bacteria every year in the United States. This problem is exacerbated by the increase in resistance to common antibiotics that are routinely used to treat these infections, creating an urgent need for innovative ways to treat and prevent virulence caused by these pathogens. Many Gram-negative pathogenic bacteria use a type III secretion system (T3SS) to inject toxins and other effector proteins directly into host cells. The T3SS has become a popular anti-virulence target because it is required for pathogenesis and knockouts have attenuated virulence. It is also not required for survival, which should result in less selective pressure for resistance formation against T3SS inhibitors. In this review, we will highlight selected examples of direct antibody immunizations and the use of antibodies in immunotherapy treatments that target the bacterial T3SS. These examples include antibodies targeting the T3SS of Pseudomonas aeruginosa, Yersinia pestis, Escherichia coli, Salmonella enterica, Shigella spp., and Chlamydia trachomatis.

PMID: 32726928 [PubMed]

Small hydrophobic proteins, encoded by small open reading frames, contribute to the adaptation of bacterial pathogens to environmental stresses, often through the interaction with larger membrane proteins, and can thus regulate bacterial virulence. Identification and functional characterization of small membrane proteins, especially negative regulators of virulence factors or toxic proteins, paves the way for the discovery of synthetic peptides with antivirulence or antibacterial activity.

Abstract

Bacterial small proteins (below 50 amino acids) encoded by small open reading frames (sORFs) are recognized as an emerging class of functional molecules that have been largely overlooked in the past. While some were uncovered serendipitously, global approaches have recently been developed to detect these sORFs. A large portion of small proteins appears to be hydrophobic and located in the bacterial membrane. In the present review, we describe functional small hydrophobic proteins discovered in pathogenic bacteria and report recent advances in the discovery of additional ones. Small membrane proteins contribute to bacterial adaptation to changing environments and often appear to be implicated in negative feedback regulation loops by modulating the function or stability of larger membrane proteins. A subset of these proteins belongs to toxin‐antitoxin modules. We highlight the features of characterized hydrophobic small proteins that may pave the way for identification of the functional small proteins among novel sORFs discovered. Besides providing new insights into bacterial pathogenesis, identification of naturally occurring small hydrophobic proteins of pathogenic bacteria can lead to new therapeutic interventions, as recently shown with the development of synthetic peptides derived from natural small proteins that display antibacterial or antivirulence properties.