Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography-Native Mass Spectrometry.

The characterization of proteins and complexes in biological systems is essential to establish their critical properties and to understand their unique functions in a plethora of bioprocesses. However, it is highly difficult to analyze low levels of intact proteins in their native states (especially those exceeding 30 kDa) with liquid chromatography (LC)-mass spectrometry (MS). Herein, we describe for the first time the use of nanoflow ion-exchange chromatography directly coupled with native MS to resolve mixtures of intact proteins. Reference proteins and protein complexes with molecular weights between 10 and 150 kDa and a model cell lysate were separated using a salt-mediated pH gradient method with volatile additives. The method allowed for low detection limits (0.22 pmol of monoclonal antibodies), while proteins presented nondenatured MS (low number of charges and limited charge state distributions), and the oligomeric state of the complexes analyzed was mostly kept. Excellent chromatographic separations including the resolution of different proteoforms of large proteins (>140 kDa) and a peak capacity of 82 in a 30 min gradient were obtained. The proposed setup and workflows show great potential for analyzing diverse proteoforms in native top-down proteomics, opening unprecedented opportunities for clinical studies and other sample-limited applications.

Automated sample preparation with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate and iodoacetamide derivatization reagents for enantioselective liquid chromatography tandem mass spectrometry amino acid analysis.

Enantioselective amino acid analysis is gaining increasing importance in pharmaceutical, biomedical and food sciences. While there are many methods available for enantiomer separation of amino acids, the simultaneous analysis of all chiral proteinogenic amino acids by a single method with one column and a single condition is still challenging. Herein, we report an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS) assay using Chiralpak QN-AX as chiral column. With 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) as derivatization reagent, efficient enantioselective separation of D- and L-amino acids using HPLC has become possible. Thiol-containing amino acids like Cys are alkylated prior to AQC-labelling. A protocol for automated sample preparation including both derivatization step and calibrator preparation is presented. For compensating matrix effects, u-13C15N-labelled internal standards (IS) were employed. The method was validated and applied to the enantioselective analysis of amino acids in a bacterial fermentation broth.

Disrupting iron homeostasis can potentiate colistin activity and overcome colistin resistance mechanisms in Gram-Negative Bacteria.

Acinetobacter baumannii is a Gram-negative priority pathogen that can readily overcome antibiotic treatment through a range of intrinsic and acquired resistance mechanisms. Treatment of carbapenem-resistant A. baumannii largely relies on the use of colistin in cases where other treatment options have been exhausted. However, the emergence of resistance against this last-line drug has significantly increased amongst clinical strains. In this study, we identify the phytochemical kaempferol as a potentiator of colistin activity. When administered singularly, kaempferol has no effect on growth but does impact biofilm formation. Nonetheless, co-administration of kaempferol with sub-inhibitory concentrations of colistin exposes bacteria to a metabolic Achilles heel, whereby kaempferol-induced dysregulation of iron homeostasis leads to bacterial killing. We demonstrate that this effect is due to the disruption of Fenton's reaction, and therefore to a lethal build-up of toxic reactive oxygen species in the cell. Furthermore, we show that this vulnerability can be exploited to overcome both intrinsic and acquired colistin resistance in clinical strains of A. baumannii and E. coli in vitro and in the Galleria mellonella model of infection. Overall, our findings provide a proof-of-principle demonstration that targeting iron homeostasis is a promising strategy for enhancing the efficacy of colistin and overcoming colistin-resistant infections.

Antibiotic potentiation and inhibition of cross-resistance in pathogens associated with cystic fibrosis.

Critical Gram-negative pathogens, like Pseudomonas, Stenotrophomonas and Burkholderia, have become resistant to most antibiotics. Complex resistance profiles together with synergistic interactions between these organisms increase the likelihood of treatment failure in distinct infection settings, for example in the lungs of cystic fibrosis patients. Here, we discover that cell envelope protein homeostasis pathways underpin both antibiotic resistance and cross-protection in CF-associated bacteria. We find that inhibition of oxidative protein folding inactivates multiple species-specific resistance proteins. Using this strategy, we sensitize multi-drug resistant Pseudomonas aeruginosa to ß-lactam antibiotics and demonstrate promise of new treatment avenues for the recalcitrant pathogen Stenotrophomonas maltophilia. The same approach also inhibits cross-protection between resistant S. maltophilia and susceptible P. aeruginosa, allowing eradication of both commonly co-occurring CF-associated organisms. Our results provide the basis for the development of next-generation strategies that target antibiotic resistance, while also impairing specific interbacterial interactions that enhance the severity of polymicrobial infections.

Making a chink in their armor: Current and next-generation antimicrobial strategies against the bacterial cell envelope.

Gram-negative bacteria are uniquely equipped to defeat antibiotics. Their outermost layer, the cell envelope, is a natural permeability barrier that contains an array of resistance proteins capable of neutralizing most existing antimicrobials. As a result, its presence creates a major obstacle for the treatment of resistant infections and for the development of new antibiotics. Despite this seemingly impenetrable armor, in-depth understanding of the cell envelope, including structural, functional and systems biology insights, has promoted efforts to target it that can ultimately lead to the generation of new antibacterial therapies. In this article, we broadly overview the biology of the cell envelope and highlight attempts and successes in generating inhibitors that impair its function or biogenesis. We argue that the very structure that has hampered antibiotic discovery for decades has untapped potential for the design of novel next-generation therapeutics against bacterial pathogens.

Carbapenem-Resistant and ESBL-Producing Enterobacterales Emerging in Central Texas.

Purpose: Carbapenem-resistant Enterobacterales (CRE) are subject to intense global monitoring in an attempt to maintain awareness of prevalent and emerging resistance mechanisms and to inform treatment and infection prevention strategies. CRE and extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales are not usually examined collectively in regards to their shared pool of resistance determinants. Here, we genetically and phenotypically assess clinical isolates of CRE and extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales in the growing region of Central Texas, where CRE are emergent and occurrence of non-carbapenemase-producing-CRE (non-CP-CRE) infections is increasing. Methods: CRE (n=16) and ESBL-producing Enterobacterales (n=116) isolates were acquired from a regional hospital in Central Texas between December 2018 and January 2020. Isolates were assessed genetically and phenotypically using antibiotic susceptibility testing, targeted PCR, and whole genome sequencing. Results: CRE infections are increasing in incidence in Central Texas, and Klebsiella pneumoniae is causing the majority of these infections. Moreover, K. pneumoniae sequence type (ST) 307 is commonly found among both non-CP-CRE and EBSL-producing strains. Isolates carry similar plasmids harboring the gene for the ESBL CTX-M-15 and belong to the global lineage, rather than the Texas lineage, of ST307. Antibiotic resistance profiles, sequence data, and clinical records suggest that porin mutations may promote the transition of ST307 isolates from ESBL-producing to non-CP-CRE. In addition to antibiotic resistance mechanisms, several CRE isolates harbor active colicinogenic plasmids, which might influence the competitiveness of these bacteria during patient colonization. Conclusion: K. pneumoniae of the global ST307 lineage is circulating in Central Texas and is responsible for both non-CP CRE and ESBL-producing Enterobacterales infections. Enhanced surveillance is needed to understand the possible routes for the emergence of non-CP-CRE from EBSL-producing strains.

Designing and identifying ß-hairpin peptide macrocycles with antibiotic potential.

Peptide macrocycles are a rapidly emerging class of therapeutic, yet the design of their structure and activity remains challenging. This is especially true for those with ß-hairpin structure due to weak folding properties and a propensity for aggregation. Here, we use proteomic analysis and common antimicrobial features to design a large peptide library with macrocyclic ß-hairpin structure. Using an activity-driven high-throughput screen, we identify dozens of peptides killing bacteria through selective membrane disruption and analyze their biochemical features via machine learning. Active peptides contain a unique constrained structure and are highly enriched for cationic charge with arginine in their turn region. Our results provide a synthetic strategy for structured macrocyclic peptide design and discovery while also elucidating characteristics important for ß-hairpin antimicrobial peptide activity.

The biogenesis of ß-lactamase enzymes.

The discovery of penicillin by Alexander Fleming marked a new era for modern medicine, allowing not only the treatment of infectious diseases, but also the safe performance of life-saving interventions, like surgery and chemotherapy. Unfortunately, resistance against penicillin, as well as more complex ß-lactam antibiotics, has rapidly emerged since the introduction of these drugs in the clinic, and is largely driven by a single type of extra-cytoplasmic proteins, hydrolytic enzymes called ß-lactamases. While the structures, biochemistry and epidemiology of these resistance determinants have been extensively characterized, their biogenesis, a complex process including multiple steps and involving several fundamental biochemical pathways, is rarely discussed. In this review, we provide a comprehensive overview of the journey of ß-lactamases, from the moment they exit the ribosomal channel until they reach their final cellular destination as folded and active enzymes.

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding.

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here, we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse ß-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.

Antibiotics, like penicillin, are the foundation of modern medicine, but bacteria are evolving to resist their effects. Some of the most harmful pathogens belong to a group called the 'Gram-negative bacteria', which have an outer layer ­ called the cell envelope ­ that acts as a drug barrier. This envelope contains antibiotic resistance proteins that can deactivate or repel antibiotics or even pump them out of the cell once they get in. One way to tackle antibiotic resistance could be to stop these proteins from working. Proteins are long chains of building blocks called amino acids that fold into specific shapes. In order for a protein to perform its role correctly, it must fold in the right way. In bacteria, a protein called DsbA helps other proteins fold correctly by holding them in place and inserting links called disulfide bonds. It was unclear whether DsbA plays a role in the folding of antibiotic resistance proteins, but if it did, it might open up new ways to treat antibiotic resistant infections. To find out more, Furniss, Kaderabkova et al. collected the genes that code for several antibiotic resistance proteins and put them into Escherichia coli bacteria, which made the bacteria resistant to antibiotics. Furniss, Kaderabkova et al. then stopped the modified E. coli from making DsbA, which led to the antibiotic resistance proteins becoming unstable and breaking down because they could not fold correctly. Further experiments showed that blocking DsbA with a chemical inhibitor in other pathogenic species of Gram-negative bacteria made these bacteria more sensitive to antibiotics that they would normally resist. To demonstrate that using this approach could work to stop infections by these bacteria, Furniss, Kaderabkova et al. used Gram-negative bacteria that produced antibiotic resistance proteins but could not make DsbA to infect insect larvae. The larvae were then treated with antibiotics, which increased their survival rate, indicating that blocking DsbA may be a good approach to tackling antibiotic resistant bacteria. According to the World Health Organization, developing new treatments against Gram-negative bacteria is of critical importance, but the discovery of new drugs has ground to a halt. One way around this is to develop ways to make existing drugs work better. Making drugs that block DsbA could offer a way to treat resistant infections using existing antibiotics in the future.

Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic.

Colistin is a polymyxin antibiotic of last resort for the treatment of infections caused by multi-drug-resistant Gram-negative bacteria. By targeting lipopolysaccharide (LPS), the antibiotic disrupts both the outer and cytoplasmic membranes, leading to bacterial death and lysis. Colistin resistance in Escherichia coli occurs via mutations in the chromosome or the acquisition of mobilized colistin-resistance (mcr) genes. Both these colistin-resistance mechanisms result in chemical modifications to the LPS, with positively charged moieties added at the cytoplasmic membrane before the LPS is transported to the outer membrane. We have previously shown that MCR-1-mediated LPS modification protects the cytoplasmic but not the outer membrane from damage caused by colistin, enabling bacterial survival. However, it remains unclear whether this observation extends to colistin resistance conferred by other mcr genes, or resistance due to chromosomal mutations. Using a panel of clinical E. coli that had acquired mcr -1, -1.5, -2, -3, -3.2 or -5, or had acquired polymyxin resistance independently of mcr genes, we found that almost all isolates were susceptible to colistin-mediated permeabilization of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilization of the outer membrane of colistin-resistant isolates by the polymyxin is in turn sufficient to sensitize bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in these E. coli isolates is due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of the mechanism of resistance.

Identification of Tse8 as a Type VI secretion system toxin from Pseudomonas aeruginosa that targets the bacterial transamidosome to inhibit protein synthesis in prey cells.

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion-site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis and validate the use of a transposon directed insertion site sequencing-based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria.

The Breadth and Molecular Basis of Hcp-Driven Type VI Secretion System Effector Delivery.

The type VI secretion system (T6SS) is a bacterial nanoscale weapon that delivers toxins into prey ranging from bacteria and fungi to animal hosts. The cytosolic contractile sheath of the system wraps around stacked hexameric rings of Hcp proteins, which form an inner tube. At the tip of this tube is a puncturing device comprising a trimeric VgrG topped by a monomeric PAAR protein. The number of toxins a single system delivers per firing event remains unknown, since effectors can be loaded on diverse sites of the T6SS apparatus, notably the inner tube and the puncturing device. Each VgrG or PAAR can bind one effector, and additional effector cargoes can be carried in the Hcp ring lumen. While many VgrG- and PAAR-bound toxins have been characterized, to date, very few Hcp-bound effectors are known. Here, we used 3 known Pseudomonas aeruginosa Hcp proteins (Hcp1 to -3), each of which associates with one of the three T6SSs in this organism (H1-T6SS, H2-T6SS, and H3-T6SS), to perform in vivo pulldown assays. We confirmed the known interactions of Hcp1 with Tse1 to -4, further copurified a Hcp1-Tse4 complex, and identified potential novel Hcp1-bound effectors. Moreover, we demonstrated that Hcp2 and Hcp3 can shuttle T6SS cargoes toxic to Escherichia coli. Finally, we used a Tse1-Bla chimera to probe the loading strategy for Hcp passengers and found that while large effectors can be loaded onto Hcp, the formed complex jams the system, abrogating T6SS function. IMPORTANCE The type VI secretion system (T6SS) is an effective weapon used by bacteria to outgrow or kill competitors. It can be used by endogenous commensal microbiota to prevent invasion by pathogens or by pathogens to overcome resident flora and successfully colonize a host or a specific environmental niche. The T6SS is a key contributor to this continuous arms race between organisms as it delivers a multitude of toxins directed at essential processes, such as nucleic acid synthesis and replication, cell wall and membrane integrity, protein synthesis, or cofactor abundance. Many T6SS toxins with unknown function remain to be discovered, whose yet-uncharacterized targets could be exploited for antimicrobial drug design. The systematic search for these toxins is not facilitated by the presence of readily recognizable T6SS motifs, and unbiased screening approaches are thus required. Here, we successfully used a known shuttle for cargo T6SS effectors, Hcp, as bait to identify uncharacterized toxins.

Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane.

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

Antibiotics are life-saving medicines, but many bacteria now have the ability to resist their effects. For some infections, all frontline antibiotics are now ineffective. To treat infections caused by these highly resistant bacteria, clinicians must use so-called 'antibiotics of last resort'. These antibiotics include a drug called colistin, which is moderately effective, but often fails to eradicate the infection. One of the challenges to making colistin more effective is that its mechanism is poorly understood. Bacteria have two layers of protection against the outside world: an outer cell membrane and an inner cell membrane. To kill them, colistin must punch holes in both. First, it disrupts the outer membrane by interacting with molecules called lipopolysaccharides. But how it disrupts the inner membrane was unclear. Bacteria have evolved several different mechanisms that make them resistant to the effects of colistin. Sabnis et al. reasoned that understanding how these mechanisms protected bacteria could reveal how the antibiotic works to damage the inner cell membrane. Sabnis et al. examined the effects of colistin on Escherichia coli bacteria with and without resistance to the antibiotic. Exposing these bacteria to colistin revealed that the antibiotic damages both layers of the cell surface in the same way, targeting lipopolysaccharide in the inner membrane as well as the outer membrane. Next, Sabnis et al. used this new information to make colistin work better. They found that the effects of colistin were magnified when it was combined with the experimental antibiotic murepavadin, which caused lipopolysaccharide to build up at the inner membrane. This allowed colistin to punch more holes through the inner membrane, making colistin more effective at killing bacteria. To find out whether this combination of colistin and murepavadin could work as a clinical treatment, Sabnis et al. tested it on mice with Pseudomonas aeruginosa infections in their lungs. Colistin was much better at killing Pseudomonas aeruginosa and treating infections when combined with murepavadin than it was on its own. Pseudomonas aeruginosa bacteria can cause infections in the lungs of people with cystic fibrosis. At the moment, patients receive colistin in an inhaled form to treat these infections, but it is not always successful. The second drug used in this study, murepavadin, is about to enter clinical trials as an inhaled treatment for lung infections too. If the trial is successful, it may be possible to use both drugs in combination to treat lung infections in people with cystic fibrosis.

A novel stabilization mechanism for the type VI secretion system sheath.

The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short form (TssAS) and a long form (TssAL). While TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. Here we discover a class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for some time or fire immediately after sheath extension. We propose that this diversity in firing dynamics could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.

Droplet printing reveals the importance of micron-scale structure for bacterial ecology.

Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology. However, a test of this hypothesis requires manipulation at the fine scales at which spatial structure naturally occurs. Here we develop a droplet-based printing method to arrange bacterial genotypes across a sub-millimetre array. We print strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins. Our experiments reveal that toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge where susceptible strains can persist in large numbers. Moving to competitions between toxin producers reveals that spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between competing strains to understand how ecological refuges form, which shows that cells closest to a toxin producer mop up the toxin and protect their clonemates. Our work provides a method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in ecology.

Harnessing the potential of bacterial oxidative folding to aid protein production.

Protein folding is central to both biological function and recombinant protein production. In bacterial expression systems, which are easy to use and offer high protein yields, production of the protein of interest in its native fold can be hampered by the limitations of endogenous posttranslational modification systems. Disulfide bond formation, entailing the covalent linkage of proximal cysteine amino acids, is a fundamental posttranslational modification reaction that often underpins protein stability, especially in extracytoplasmic environments. When these bonds are not formed correctly, the yield and activity of the resultant protein are dramatically decreased. Although the mechanism of oxidative protein folding is well understood, unwanted or incorrect disulfide bond formation often presents a stumbling block for the expression of cysteine-containing proteins in bacteria. It is therefore important to consider the biochemistry of prokaryotic disulfide bond formation systems in the context of protein production, in order to take advantage of the full potential of such pathways in biotechnology applications. Here, we provide a critical overview of the use of bacterial oxidative folding in protein production so far, and propose a practical decision-making workflow for exploiting disulfide bond formation for the expression of any given protein of interest.

Staphylococcal DNA Repair Is Required for Infection.

To cause infection, Staphylococcus aureus must withstand damage caused by host immune defenses. However, the mechanisms by which staphylococcal DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as being important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double-strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double-strand breaks through reactive oxygen species (ROS) generated by the respiratory burst, which are repaired by RexAB, leading to the induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted the survival of these pathogens in human blood, suggesting that DNA double-strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that the repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.IMPORTANCE To cause infection, bacteria must survive attack by the host immune system. For many bacteria, including the major human pathogen Staphylococcus aureus, the greatest threat is posed by neutrophils. These immune cells ingest the invading organisms and try to kill them with a cocktail of chemicals that includes reactive oxygen species (ROS). The ability of S. aureus to survive this attack is crucial for the progression of infection. However, it was not clear how the ROS damaged S. aureus and how the bacterium repaired this damage. In this work, we show that ROS cause breaks in the staphylococcal DNA, which must be repaired by a two-protein complex known as RexAB; otherwise, the bacterium is killed, and it cannot sustain infection. This provides information on the type of damage that neutrophils cause S. aureus and the mechanism by which this damage is repaired, enabling infection.

Local frustration determines loop opening during the catalytic cycle of an oxidoreductase.

Local structural frustration, the existence of mutually exclusive competing interactions, may explain why some proteins are dynamic while others are rigid. Frustration is thought to underpin biomolecular recognition and the flexibility of protein-binding sites. Here, we show how a small chemical modification, the oxidation of two cysteine thiols to a disulfide bond, during the catalytic cycle of the N-terminal domain of the key bacterial oxidoreductase DsbD (nDsbD), introduces frustration ultimately influencing protein function. In oxidized nDsbD, local frustration disrupts the packing of the protective cap-loop region against the active site allowing loop opening. By contrast, in reduced nDsbD the cap loop is rigid, always protecting the active-site thiols from the oxidizing environment of the periplasm. Our results point toward an intricate coupling between the dynamics of the active-site cysteines and of the cap loop which modulates the association reactions of nDsbD with its partners resulting in optimized protein function.

Detection of Colistin Resistance in Escherichia coli by Use of the MALDI Biotyper Sirius Mass Spectrometry System.

Polymyxin antibiotics are a last-line treatment for multidrug-resistant Gram-negative bacteria. However, the emergence of colistin resistance, including the spread of mobile mcr genes, necessitates the development of improved diagnostics for the detection of colistin-resistant organisms in hospital settings. The recently developed MALDIxin test enables detection of colistin resistance by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in less than 15 min but is not optimized for the mass spectrometers commonly found in clinical microbiology laboratories. In this study, we adapted the MALDIxin test for the MALDI Biotyper Sirius MALDI-TOF MS system (Bruker Daltonics). We optimized the sample preparation protocol by using a set of 6 mobile colistin resistance (MCR) protein-expressing Escherichia coli clones and validated the assay with a collection of 40 E. coli clinical isolates, including 19 confirmed MCR protein producers, 12 colistin-resistant isolates that tested negative for commonly encountered mcr genes (i.e., likely chromosomally resistant isolates), and 9 polymyxin-susceptible isolates. We calculated polymyxin resistance ratio (PRR) values from the acquired spectra; PRR values of 0, indicating polymyxin susceptibility, were obtained for all colistin-susceptible E. coli isolates, whereas positive PRR values, indicating resistance to polymyxins, were obtained for all resistant strains, independent of the genetic basis of resistance. Thus, we report a preliminary feasibility study showing that an optimized version of the MALDIxin test adapted for the routine MALDI Biotyper Sirius system provides an unbiased, fast, reliable, cost-effective, and high-throughput way of detecting colistin resistance in clinical E. coli isolates.