Nucleoid-Associated Proteins HU and IHF: Oligomerization in Solution and Hydrodynamic Properties.

Structure and function of bacterial nucleoid is controlled by the nucleoid-associated proteins (NAP). In any phase of growth, various NAPs, acting sequentially, condense nucleoid and facilitate formation of its transcriptionally active structure. However, in the late stationary phase, only one of the NAPs, Dps protein, is strongly expressed, and DNA-protein crystals are formed that transform nucleoid into a static, transcriptionally inactive structure, effectively protected from the external influences. Discovery of crystal structures in living cells and association of this phenomenon with the bacterial resistance to antibiotics has aroused great interest in studying this phenomenon. The aim of this work is to obtain and compare structures of two related NAPs (HU and IHF), since they are the ones that accumulate in the cell at the late stationary stage of growth, which precedes formation of the protective DNA-Dps crystalline complex. For structural studies, two complementary methods were used in the work: small-angle X-ray scattering (SAXS) as the main method for studying structure of proteins in solution, and dynamic light scattering as a complementary one. To interpret the SAXS data, various approaches and computer programs were used (in particular, the evaluation of structural invariants, rigid body modeling and equilibrium mixture analysis in terms of the volume fractions of its components were applied), which made it possible to determine macromolecular characteristics and obtain reliable 3D structural models of various oligomeric forms of HU and IHF proteins with ~2 nm resolution typical for SAXS. It was shown that these proteins oligomerize in solution to varying degrees, and IHF is characterized by the presence of large oligomers consisting of initial dimers arranged in a chain. An analysis of the experimental and published data made it possible to hypothesize that just before the Dps expression, it is IHF that forms toroidal structures previously observed in vivo and prepares the platform for formation of DNA-Dps crystals. The results obtained are necessary for further investigation of the phenomenon of biocrystal formation in bacterial cells and finding ways to overcome resistance of various pathogens to external conditions.

IHF and Fis as Escherichia coli Cell Cycle Regulators: Activation of the Replication Origin oriC and the Regulatory Cycle of the DnaA Initiator.

This review summarizes current knowledge about the mechanisms of timely binding and dissociation of two nucleoid proteins, IHF and Fis, which play fundamental roles in the initiation of chromosomal DNA replication in Escherichia coli. Replication is initiated from a unique replication origin called oriC and is tightly regulated so that it occurs only once per cell cycle. The timing of replication initiation at oriC is rigidly controlled by the timely binding of the initiator protein DnaA and IHF to oriC. The first part of this review presents up-to-date knowledge about the timely stabilization of oriC-IHF binding at oriC during replication initiation. Recent advances in our understanding of the genome-wide profile of cell cycle-coordinated IHF binding have revealed the oriC-specific stabilization of IHF binding by ATP-DnaA oligomers at oriC and by an initiation-specific IHF binding consensus sequence at oriC. The second part of this review summarizes the mechanism of the timely regulation of DnaA activity via the chromosomal loci DARS2 (DnaA-reactivating sequence 2) and datA. The timing of replication initiation at oriC is controlled predominantly by the phosphorylated form of the adenosine nucleotide bound to DnaA, i.e., ATP-DnaA, but not ADP-ADP, is competent for initiation. Before initiation, DARS2 increases the level of ATP-DnaA by stimulating the exchange of ADP for ATP on DnaA. This DARS2 function is activated by the site-specific and timely binding of both IHF and Fis within DARS2. After initiation, another chromosomal locus, datA, which inactivates ATP-DnaA by stimulating ATP hydrolysis, is activated by the timely binding of IHF. A recent study has shown that ATP-DnaA oligomers formed at DARS2-Fis binding sites competitively dissociate Fis via negative feedback, whereas IHF regulation at DARS2 and datA still remains to be investigated. This review summarizes the current knowledge about the specific role of IHF and Fis in the regulation of replication initiation and proposes a mechanism for the regulation of timely IHF binding and dissociation at DARS2 and datA.

A Humanized Monoclonal Antibody Potentiates Killing of Diverse Biofilm-Forming Respiratory Tract Pathogens by Antibiotics.

New strategies to treat diseases in which biofilms contribute significantly to pathogenesis are needed, as biofilm-resident bacteria are highly recalcitrant to antibiotics due to physical biofilm architecture and a canonically quiescent metabolism, among many additional attributes. We, and others, have shown that when biofilms are dispersed or disrupted, bacteria released from biofilm residence are in a distinct physiologic state that, in part, renders these bacteria highly sensitive to killing by specific antibiotics. We sought to demonstrate the breadth of the ability of a recently humanized monoclonal antibody against an essential biofilm structural element (DNABII protein) to disrupt biofilms formed by respiratory tract pathogens and potentiate antibiotic-mediated killing of bacteria released from biofilm residence. Biofilms formed by six respiratory tract pathogens were significantly disrupted by the humanized monoclonal antibody in a dose- and time-dependent manner, as corroborated by confocal laser scanning microscopy (CLSM) imaging. Bacteria newly released from the biofilms of 3 of 6 species were significantly more sensitive than their planktonic counterparts to killing by 2 of 3 antibiotics currently used clinically and were now also equally as sensitive to killing by the 3rd antibiotic. The remaining 3 pathogens were significantly more susceptible to killing by all 3 antibiotics. A humanized monoclonal antibody directed against protective epitopes of a DNABII protein effectively released six diverse respiratory tract pathogens from biofilm residence in a phenotypic state that was now as, or significantly more, sensitive to killing by three antibiotics currently indicated for use clinically. These data support this targeted, combinatorial, species-agnostic therapy to mitigate chronic bacterial diseases.

vgrG is separately transcribed from hcp in T6SS orphan clusters and is under the regulation of IHF and HapR.

V. cholerae, the causative agent of cholera epidemic, and V. fluvialis, the emerging foodborne pathogen, share highly homologous T6SS consisting of one large cluster and two small orphan or auxiliary clusters, and each of which was generally recognized as one operon. Here, we showed that the genes in each of the small clusters are organized into two transcriptional units. Specifically, the inner tube coding gene hcp/tssD is highly transcribed as one monocistron, while the tip component vgrG/tssI and its downstream effector and immunity genes are in one polycistron with very low transcriptional level. This conclusion is supported by qPCR analysis of mRNA abundance, reporter fusion analysis and transcriptional unit definition with RT-PCR analysis. Taking tssI2_a of V. fluvialis as an example, we further demonstrated that quorum sensing (QS) regulator HapR and global regulator IHF activate vgrG/tssI transcription by directly binding to its promoter region. Taken together, current studies deepen our understanding of T6SS system, highlighting its regulatory complexity during functional execution process.

Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1-Cas2 integrase.

Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro However, this nonspecific integration activity is rare in vivo Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1-Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mm spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1-Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.

Nontypeable Haemophilus influenzae releases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery.

Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and resistance to treatment of multiple human respiratory tract diseases including otitis media, chronic rhinosinusitis, and exacerbations of both cystic fibrosis and chronic obstructive pulmonary disease. Extracellular DNA (eDNA) and associated DNABII proteins are essential to the overall architecture and structural integrity of biofilms formed by NTHI and all other bacterial pathogens tested to date. Although cell lysis and outer-membrane vesicle extrusion are possible means by which these canonically intracellular components might be released into the extracellular environment for incorporation into the biofilm matrix, we hypothesized that NTHI additionally used a mechanism of active DNA release. Herein, we describe a mechanism whereby DNA and associated DNABII proteins transit from the bacterial cytoplasm to the periplasm via an inner-membrane pore complex (TraC and TraG) with homology to type IV secretion-like systems. These components exit the bacterial cell through the ComE pore through which the NTHI type IV pilus is expressed. The described mechanism is independent of explosive cell lysis or cell death, and the release of DNA is confined to a discrete subpolar location, which suggests a novel form of DNA release from viable NTHI. Identification of the mechanisms and determination of the kinetics by which critical biofilm matrix-stabilizing components are released will aid in the design of novel biofilm-targeted therapeutic and preventative strategies for diseases caused by NTHI and many other human pathogens known to integrate eDNA and DNABII proteins into their biofilm matrix.

H-NS, IHF, and DnaA lead to changes in nucleoid organizations, replication initiation, and cell division.

Histone-like nucleoid-structuring protein (H-NS) and integration host factor (IHF) are major nucleoid-associated proteins, and DnaA, a replication initiator, may also be related with nucleoid compaction. It has been shown that protein-dependent DNA compaction is related with many aspects of bacterial physiology, including transcription, DNA replication, and site-specific recombination. However, the mechanism of bacterial physiology resulting from nucleoid compaction remains unknown. Here, we show that H-NS is important for correct nucleoid compaction in a medium-independent manner. H-NS-mediated nucleoid compaction is not required for correct cell division, but the latter is dependent on H-NS in rich medium. Further, it is found that the IHFα-mediated nucleoid compaction is needed for correct cell division, and the effect is dependent on medium. Also, we show that the effects of H-NS and IHF on nucleoid compaction are cumulative. Interestingly, DnaA also plays an important role in nucleoid compaction, and the effect of DnaA on nucleoid compaction appears to be related to cell division in a medium-dependent manner. The results presented here suggest that scrambled initiation of replication, improper cell division, and slow growth is likely associated with disturbances in nucleoid organization directly or indirectly.

Initiation of DNA Replication at the Chromosomal Origin of E. coli, oriC.

The Escherichia coli chromosomal origin consists of a duplex-unwinding region and a region bearing a DNA-bending protein, IHF-binding site, and clusters of binding sites for the initiator protein DnaA. ATP-DnaA molecules form highly organized oligomers in a process stimulated by DiaA, a DnaA-binding protein. The resultant ATP-DnaA complexes promote local unwinding of oriC with the aid of IHF, for which specific interaction of DnaA with the single-stranded DNA is crucial. DnaA complexes also interact with DnaB helicases bound to DnaC loaders, promoting loading of DnaB onto the unwound DNA strands for bidirectional replication. Initiation of replication is strictly regulated during the cell cycle by multiple regulatory systems for oriC and DnaA. The activity of oriC is regulated by its methylation state, whereas that of DnaA depends on the form of the bound nucleotide. ATP-DnaA can be yielded from initiation-inactive ADP-DnaA in a timely manner depending on specific chromosomal DNA elements termed DARS (DnaA-reactivating sequences). After initiation, DnaA-bound ATP is hydrolyzed by two systems, yielding ADP-DnaA. In this review, these and other mechanisms of initiation and its regulation in E. coli are described.

Read-through transcription of tRNA underlies the cell cycle-dependent dissociation of IHF from the DnaA-inactivating sequence datA.

Timely initiation of chromosomal DNA replication in Escherichia coli is achieved by cell cycle-coordinated regulation of the replication origin, oriC, and the replication initiator, ATP-DnaA. Cellular levels of ATP-DnaA increase and peak at the time for initiation at oriC, after which hydrolysis of DnaA-bound ATP causes those to fall, yielding initiation-inactive ADP-DnaA. This hydrolysis is facilitated by the chromosomal locus datA located downstream of the tRNA-Gly (glyV-X-Y) operon, which possesses a cluster of DnaA-binding sequences and a single binding site (IBS) for the DNA bending protein IHF (integration host factor). While IHF binding activates the datA function and is regulated to occur specifically at post-initiation time, the underlying regulatory mechanisms remain obscure. Here, we demonstrate that datA-IHF binding at pre-initiation time is down-regulated depending on the read-through transcription of datA IBS initiated at the glyV-X-Y promoter. During the cell cycle, the level of read-through transcription, but not promoter activity, fluctuated in a manner inversely related to datA-IHF binding. Transcription from the glyV-X-Y promoter was predominantly interrupted at datA IBS by IHF binding. The terminator/attenuator sequence of the glyV-X-Y operon, as well as DnaA binding within datA overall, contributed to attenuation of transcription upstream of datA IBS, preserving the timely fluctuation of read-through transcription. These findings provide a mechanistic insight of tRNA transcription-dependent datA-IHF regulation, in which an unidentified factor is additionally required for the timely datA-IHF dissociation, and support the significance of datA for controlling the cell cycle progression as a connecting hub of tRNA production and replication initiation.

The effect of DNA-binding proteins on insertion sequence element transposition upstream of the bgl operon in Escherichia coli.

The bglGFB operon in Escherichia coli K-12 strain BW25113, encoding the proteins necessary for the uptake and metabolism of ß-glucosides, is normally not expressed. Insertion of either IS1 or IS5 upstream of the bgl promoter activates expression of the operon only when the cell is starving in the presence of a ß-glucoside, drastically increasing transcription and allowing the cell to survive and grow using this carbon source. Details surrounding the exact mechanism and regulation of the IS insertional event remain unclear. In this work, the role of several DNA-binding proteins in how they affect the rate of insertion upstream of bgl are examined via mutation assays and protocols measuring transcription. Both Crp and IHF exert a positive effect on insertional Bgl+ mutations when present, active, and functional in the cell. Our results characterize IHF's effect in conjunction with other mutations, show that IHF's effect on IS insertion into bgl also affects other operons, and indicate that it may exert its effect by binding to and altering the DNA conformation of IS1 and IS5 in their native locations, rather than by directly influencing transposase gene expression. In contrast, the cAMP-CRP complex acts directly upon the bgl operon by binding upstream of the promoter, presumably altering local DNA into a conformation that enhances IS insertion.

Quantitation of DNA Binding Affinity Using Tethered Particle Motion.

The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered particle motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to measure binding constants of proteins binding to DNA reliably, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This change in bead motion makes it possible to measure the DNA-binding properties of proteins. We use the bacterial protein integration host factor (IHF) and the archaeal histone HMfA as examples to show how specific binding to DNA can be measured. Moreover, we show how the end-to-end distance can provide structural insights into protein-DNA binding.

The role of integration host factor in biofilm and virulence of high-alcohol-producing Klebsiella pneumoniae.

Klebsiella pneumoniae is a well-known human nosocomial pathogen with an arsenal of virulence factors, including capsular polysaccharides (CPS), fimbriae, flagella, and lipopolysaccharides (LPS). Our previous study found that alcohol acted as an essential virulence factor for high-alcohol-producing K. pneumoniae (HiAlc Kpn). Integration host factor (IHF) is a nucleoid-associated protein that functions as a global virulence regulator in Escherichia coli. However, the regulatory role of IHF in K. pneumoniae remains unknown. In the present study, we found that deletion of ihfA or ihfB resulted in a slight defect in bacterial growth, a severe absence of biofilm formation and cytotoxicity, and a significant reduction in alcohol production. RNA sequencing differential gene expression analysis showed that compared with the wild-type control, the expression of many virulence factor genes was downregulated in ΔihfA and ΔihfB strains, such as those related to CPS (rcsA, galF, wzi, and iscR), LPS (rfbABCD), type I and type III fimbriae (fim and mrk operon), cellulose (bcs operon), iron transporter (feoABC, fhuA, fhuF, tonB, exbB, and exbD), quorum sensing (lsr operon and sdiA), type II secretion system (T2SS) and type VI secretion system (T6SS) (tssG, hcp, and gspE). Of these virulence factors, CPS, LPS, fimbriae, and cellulose are involved in biofilm formation. In addition, IHF could affect the alcohol production by regulating genes related to glucose intake (ptsG), pyruvate formate-lyase, alcohol dehydrogenase, and the tricarboxylic acid (TCA) cycle. Our data provided new insights into the importance of IHF in regulating the virulence of HiAlc Kpn. IMPORTANCE Klebsiella pneumoniae is a well-known human nosocomial pathogen that causes various infectious diseases, including urinary tract infections, hospital-acquired pneumonia, bacteremia, and liver abscesses. Our previous studies demonstrated that HiAlc Kpn mediated the development of nonalcoholic fatty liver disease by producing excess endogenous alcohol in vivo. However, the regulators regulating the expression of genes related to metabolism, biofilm formation, and virulence of HiAlc Kpn remain unclear. In this study, the regulator IHF was found to positively regulate biofilm formation and many virulence factors including CPS, LPS, type I and type III fimbriae, cellulose, iron transporter, AI-2 quorum sensing, T2SS, and T6SS in HiAlc Kpn. Furthermore, IHF positively regulated alcohol production in HiAlc Kpn. Our results suggested that IHF could be a potential drug target for treating various infectious diseases caused by K. pneumoniae. Hence, the regulation of different virulence factors by IHF in K. pneumoniae requires further investigation.

Pangenome-level analysis of nucleoid-associated proteins in the Acidithiobacillia class: insights into their functional roles in mobile genetic elements biology.

Mobile genetic elements (MGEs) are relevant agents in bacterial adaptation and evolutionary diversification. Stable appropriation of these DNA elements depends on host factors, among which are the nucleoid-associated proteins (NAPs). NAPs are highly abundant proteins that bind and bend DNA, altering its topology and folding, thus affecting all known cellular DNA processes from replication to expression. Even though NAP coding genes are found in most prokaryotic genomes, their functions in host chromosome biology and xenogeneic silencing are only known for a few NAP families. Less is known about the occurrence, abundance, and roles of MGE-encoded NAPs in foreign elements establishment and mobility. In this study, we used a combination of comparative genomics and phylogenetic strategies to gain insights into the diversity, distribution, and functional roles of NAPs within the class Acidithiobacillia with a special focus on their role in MGE biology. Acidithiobacillia class members are aerobic, chemolithoautotrophic, acidophilic sulfur-oxidizers, encompassing substantial genotypic diversity attributable to MGEs. Our search for NAP protein families (PFs) in more than 90 genomes of the different species that conform the class, revealed the presence of 1,197 proteins pertaining to 12 different NAP families, with differential occurrence and conservation across species. Pangenome-level analysis revealed 6 core NAP PFs that were highly conserved across the class, some of which also existed as variant forms of scattered occurrence, in addition to NAPs of taxa-restricted distribution. Core NAPs identified are reckoned as essential based on the conservation of genomic context and phylogenetic signals. In turn, various highly diversified NAPs pertaining to the flexible gene complement of the class, were found to be encoded in known plasmids or, larger integrated MGEs or, present in genomic loci associated with MGE-hallmark genes, pointing to their role in the stabilization/maintenance of these elements in strains and species with larger genomes. Both core and flexible NAPs identified proved valuable as markers, the former accurately recapitulating the phylogeny of the class, and the later, as seed in the bioinformatic identification of novel episomal and integrated mobile elements.

Mycobacterial IHF is a highly dynamic nucleoid-associated protein that assists HupB in organizing chromatin.

Nucleoid-associated proteins (NAPs) crucially contribute to organizing bacterial chromatin and regulating gene expression. Among the most highly expressed NAPs are the HU and integration host factor (IHF) proteins, whose functional homologues, HupB and mycobacterial integration host factor (mIHF), are found in mycobacteria. Despite their importance for the pathogenicity and/or survival of tubercle bacilli, the role of these proteins in mycobacterial chromosome organization remains unknown. Here, we used various approaches, including super-resolution microscopy, to perform a comprehensive analysis of the roles of HupB and mIHF in chromosome organization. We report that HupB is a structural agent that maintains chromosome integrity on a local scale, and that the lack of this protein alters chromosome morphology. In contrast, mIHF is a highly dynamic protein that binds DNA only transiently, exhibits susceptibility to the chromosomal DNA topology changes and whose depletion leads to the growth arrest of tubercle bacilli. Additionally, we have shown that depletion of Mycobacterium smegmatis integration host factor (msIHF) leads to chromosome shrinkage and replication inhibition.

The Role of Integration Host Factor in Escherichia coli Persister Formation.

Persisters represent a small subpopulation of cells that are tolerant of killing by antibiotics and are implicated in the recalcitrance of chronic infections to antibiotic therapy. One general theme has emerged regarding persisters formed by different bacterial species, namely, a state of relative dormancy characterized by diminished activity of antibiotic targets. Within this framework, a number of studies have linked persister formation to stochastic decreases in energy-generating components, leading to low ATP and target activity. In this study, we screen knockouts in the main global regulators of Escherichia coli for their effect on persisters. A knockout in integration host factor (IHF) had elevated ATP and a diminished level of persisters. This was accompanied by an overexpression of isocitrate dehydrogenase (Icd) and a downregulation of isocitrate lyase (AceA), two genes located at the bifurcation between the tricarboxylic acid (TCA) cycle and the glyoxylate bypass. Using a translational ihfA-mVenus fusion, we sort out rare bright cells, and this subpopulation is enriched in persisters. Our results suggest that noise in the expression of ihf produces rare cells with low Icd/high AceA, diverting substrates into the glyoxylate bypass, which decreases ATP, leading to antibiotic-tolerant persisters. We further examine noise in a simple model, the lac operon, and show that a knockout of the lacI repressor increases expression of the operon and decreases persister formation. Our results suggest that noise quenching by overexpression serves as a general approach to determine the nature of persister genes in a variety of bacterial species and conditions. IMPORTANCE Persisters are phenotypic variants that survive exposure to antibiotics through temporary dormancy. Mutants with increased levels of persisters have been identified in clinical isolates, and evidence suggests these cells contribute to chronic infections and antibiotic treatment failure. Understanding the underlying mechanism of persister formation and tolerance is important for developing therapeutic approaches to treat chronic infections. In this study, we examine a global regulator, IHF, that plays a role in persister formation. We find that noise in expression of IHF contributes to persister formation, likely by regulating the switch between the TCA cycle that efficiently produces energy and the glyoxylate bypass. We extend this study to a simple model lac operon and show that when grown on lactose as the sole carbon source, noise in its expression influences ATP levels and determines persister formation. This noise is quenched by overexpression of the lac operon, providing a simple approach to test the involvement of a gene in persister formation.

Monoclonal antibodies that target extracellular DNABII proteins or the type IV pilus of nontypeable Haemophilus influenzae (NTHI) worked additively to disrupt 2-genera biofilms.

The biofilm state is the preferred lifestyle of bacteria in nature. Within a biofilm, the resident bacteria are protected from environmental stresses, antibiotics and other antimicrobials, including those due to multiple immune effectors of their host during conditions of disease. Thereby, biofilms contribute significantly to pathogenicity, recalcitrance to clearance and chronicity/recurrence of bacterial diseases, including diseases of the respiratory tract. In the absence of highly effective, biofilm-targeted therapeutics, antibiotics are commonly prescribed to attempt to treat these diseases, however, in light of the canonical resistance of biofilm-resident bacteria to antibiotic-mediated killing, this ineffectual practice often fails to resolve the diseased condition and contributes significantly to the global threat of rising antimicrobial resistance. Nontypeable Haemophilus influenzae is a common respiratory tract disease co-pathogen, often present in partnership with other airway pathogens. Herein we aspired to determine whether either of two monoclonal antibodies we developed, one specific for NTHI [directed against the majority subunit (PilA) of the type IV pilus (T4P) of NTHI] and the other able to act agnostically on all bacteria tested to date (directed against a structural protein of the biofilm matrix, a DNABII protein), were able to disrupt 2-genera biofilms wherein NTHI co-partnered with another respiratory tract pathogen. These monoclonals were tested singly as well as when within an antibody cocktail. The monoclonal directed against the NTHI antigen PilA was only effective on single species NTHI biofilms and not on single species biofilms formed by other unrelated species. However, when NTHI co-partnered with any of 5 respiratory tract pathogens tested here (Burkholderia cenocepacia, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae or Moraxella catarrhalis), this exclusively NTHI-directed monoclonal was able to disrupt these 2-genera biofilms. Conversely, the monoclonal antibody directed against protective epitopes of a DNABII protein, significantly disrupted all single species and 2-genera biofilms, which reflected the universal presence of this structural protein in all tested biofilm matrices. However, greatest release of both pathogens from a 2-genera biofilm was uniformly achieved by incubation with a 1:1 cocktail of both monoclonals. These data support the use of an approach wherein patients with respiratory tract disease could be treated with a therapeutic monoclonal antibody cocktail to release NTHI and its common co-pathogens from the protective biofilm to be killed by either traditional antibiotics and/or host immune effectors.

Crumbling the Castle: Targeting DNABII Proteins for Collapsing Bacterial Biofilms as a Therapeutic Approach to Treat Disease and Combat Antimicrobial Resistance.

Antimicrobial resistance (AMR) is a concerning global threat that, if not addressed, could lead to increases in morbidity and mortality, coupled with societal and financial burdens. The emergence of AMR bacteria can be attributed, in part, to the decreased development of new antibiotics, increased misuse and overuse of existing antibiotics, and inadequate treatment options for biofilms formed during bacterial infections. Biofilms are complex microbiomes enshrouded in a self-produced extracellular polymeric substance (EPS) that is a primary defense mechanism of the resident microorganisms against antimicrobial agents and the host immune system. In addition to the physical protective EPS barrier, biofilm-resident bacteria exhibit tolerance mechanisms enabling persistence and the establishment of recurrent infections. As current antibiotics and therapeutics are becoming less effective in combating AMR, new innovative technologies are needed to address the growing AMR threat. This perspective article highlights such a product, CMTX-101, a humanized monoclonal antibody that targets a universal component of bacterial biofilms, leading to pathogen-agnostic rapid biofilm collapse and engaging three modes of action-the sensitization of bacteria to antibiotics, host immune enablement, and the suppression of site-specific tissue inflammation. CMTX-101 is a new tool used to enhance the effectiveness of existing, relatively inexpensive first-line antibiotics to fight infections while promoting antimicrobial stewardship.

Reciprocally rewiring and repositioning the Integration Host Factor (IHF) subunit genes in Salmonella enterica serovar Typhimurium: impacts on physiology and virulence.

The Integration Host Factor (IHF) is a heterodimeric nucleoid-associated protein that plays roles in bacterial nucleoid architecture and genome-wide gene regulation. The ihfA and ihfB genes encode the subunits and are located 350 kbp apart, in the Right replichore of the Salmonella chromosome. IHF is composed of one IhfA and one IhfB subunit. Despite this 1 : 1 stoichiometry, MS revealed that IhfB is produced in 2-fold excess over IhfA. We re-engineered Salmonella to exchange reciprocally the protein-coding regions of ihfA and ihfB, such that each relocated protein-encoding region was driven by the expression signals of the other's gene. MS showed that in this 'rewired' strain, IhfA is produced in excess over IhfB, correlating with enhanced stability of the hybrid ihfB-ihfA mRNA that was expressed from the ihfB promoter. Nevertheless, the rewired strain grew at a similar rate to the wild-type and was similar in competitive fitness. However, compared to the wild-type, it was less motile, had growth-phase-specific reductions in SPI-1 and SPI-2 gene expression, and was engulfed at a higher rate by RAW macrophage. Our data show that while exchanging the physical locations of its ihf genes and the rewiring of their regulatory circuitry are well tolerated in Salmonella, genes involved in the production of type 3 secretion systems exhibit dysregulation accompanied by altered phenotypes.

Regulation of Escherichia coli Group 2 Capsule Gene Expression: A Mini Review and Update.

The expression of a group 2 capsule (K antigen), such as the K1 or K5 antigen, is a key virulence factor of Escherichia coli responsible for extra-intestinal infections. Capsule expression confers resistance to innate host defenses and plays a critical role in invasive disease. Capsule expression is temperature-dependent being expressed at 37°C but not at 20°C when outside the host. Group 2 capsule gene expression involves two convergent promoters PR1 and PR3, the regulation of which is critical to capsule expression. Temperature-dependent expression is controlled at transcriptional level directly by the binding of H-NS to PR1 and PR3 and indirectly through BipA with additional input from IHF and SlyA. More recently, other regulatory proteins, FNR, Fur, IHF, MprA, and LrhA, have been implicated in regulating capsule gene expression in response to other environmental stimuli and there is merging data for the growth phase-dependent regulation of the PR1 and PR3 promoters. The aim of the present Mini Review is to provide a unified update on the latest data on how the expression of group 2 capsules is regulated in response to a number of stimuli and the growth phase something that has not to date been addressed.

Angiopoietins, vascular endothelial growth factors and secretory phospholipase A2 in heart failure patients with preserved ejection fraction.

BACKGROUND: Heart failure (HF) is a growing public health burden, with high prevalence and mortality rates. A proportion of patients with HF have a normal ventricular ejection fraction (EF), referred to as HF with preserved EF (HFpEF), as opposed to patients with HF with reduced ejection fraction (HFrEF). HFpEF currently accounts for about 50% of all HF patients, and its prevalence is rising. Angiopoietins (ANGPTs), vascular endothelial growth factors (VEGFs) and secretory phospholipases A2 (sPLA2s) are proinflammatory mediators and key regulators of endothelial cells. METHODS: The aim of this study was to analyze the plasma concentrations of angiogenic (ANGPT1, ANGPT2, VEGF-A) and lymphangiogenic (VEGF-C, VEGF-D) factors and the plasma activity of sPLA2 in patients with HFpEF and HFrEF compared to healthy controls. RESULTS: The concentration of ANGPT1 was reduced in HFrEF compared to HFpEF patients and healthy controls. ANGPT2 levels were increased in both HFrEF and HFpEF subjects compared to controls. The ANGPT2/ANGPT1 ratio was increased in HFrEF patients compared to controls. The concentrations of both VEGF-A and VEGF-C did not differ among the three groups examined. VEGF-D was increased in both HFrEF and HFpEF patients compared to controls. Plasma activity of sPLA2 was increased in HFrEF but not in HFpEF patients compared to controls. CONCLUSIONS: Our results indicate that three different classes of proinflammatory regulators of vascular permeability and smoldering inflammation are selectively altered in HFrEF or HFpEF patients. Studies involving larger cohorts of these patients will be necessary to demonstrate the clinical implications of our findings.