Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody.

Studying the phenomenon of cellular senescence has been hindered by the lack of senescence-specific markers. As such, detection of proteins informally associated with senescence accompanies the use of senescence-associated ß-galactosidase as a collection of semiselective markers to monitor the presence of senescent cells. To identify novel biomarkers of senescence, we immunized BALB/c mice with senescent mouse lung fibroblasts and screened for antibodies that recognized senescence-associated cell-surface antigens by FACS analysis and a newly developed cell-based ELISA. The majority of antibodies that we isolated, cloned, and sequenced belonged to the IgM isotype of the innate immune system. In-depth characterization of one of these monoclonal, polyreactive natural antibodies, the IgM clone 9H4, revealed its ability to recognize the intermediate filament vimentin. By using 9H4, we observed that senescent primary human fibroblasts express vimentin on their cell surface, and MS analysis revealed a posttranslational modification on cysteine 328 (C328) by the oxidative adduct malondialdehyde (MDA). Moreover, elevated levels of secreted MDA-modified vimentin were detected in the plasma of aged senescence-accelerated mouse prone 8 mice, which are known to have deregulated reactive oxygen species metabolism and accelerated aging. Based on these findings, we hypothesize that humoral innate immunity may recognize senescent cells by the presence of membrane-bound MDA-vimentin, presumably as part of a senescence eradication mechanism that may become impaired with age and result in senescent cell accumulation.

Phospholipase A1 modulates the cell envelope phospholipid content of Brucella melitensis, contributing to polymyxin resistance and pathogenicity.

A subset of bacterial pathogens, including the zoonotic Brucella species, are highly resistant against polymyxin antibiotics. Bacterial polymyxin resistance has been attributed primarily to the modification of lipopolysaccharide; however, it is unknown what additional mechanisms mediate high-level resistance against this class of drugs. This work identified a role for the Brucella melitensis gene bveA (BMEII0681), encoding a predicted esterase, in the resistance of B. melitensis to polymyxin B. Characterization of the enzymatic activity of BveA demonstrated that it is a phospholipase A1 with specificity for phosphatidylethanolamine (PE). Further, lipidomic analysis of B. melitensis revealed an excess of PE lipids in the bacterial membranes isolated from the bveA mutant. These results suggest that by lowering the PE content of the cell envelope, BveA increases the resistance of B. melitensis to polymyxin B. BveA was required for survival and replication of B. melitensis in macrophages and for persistent infection in mice. BveA family esterases are encoded in the genomes of the alphaproteobacterial species that coexist with the polymyxin-producing bacteria in the rhizosphere, suggesting that maintenance of a low PE content in the bacterial cell envelope may be a shared persistence strategy for association with plant and mammalian hosts.

Genetic pathway in acquisition and loss of vancomycin resistance in a methicillin resistant Staphylococcus aureus (MRSA) strain of clonal type USA300.

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible "parental" strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a "stealth" strategy to evade detection by the host immune system.

genetic determinants of intrinsic colistin tolerance in Acinetobacter baumannii.

Acinetobacter baumannii is a leading cause of multidrug-resistant infections worldwide. This organism poses a particular challenge due to its ability to acquire resistance to new antibiotics through adaptation or mutation. This study was undertaken to determine the mechanisms governing the adaptability of A. baumannii to the antibiotic colistin. Screening of a transposon mutant library identified over 30 genes involved in inducible colistin resistance in A. baumannii. One of the genes identified was lpsB, which encodes a glycosyltransferase involved in lipopolysaccharide (LPS) synthesis. We demonstrate that loss of LpsB function results in increased sensitivity to both colistin and cationic antimicrobial peptides of the innate immune system. Moreover, LpsB is critical for pathogenesis in a pulmonary model of infection. Taken together, these data define bacterial processes required for intrinsic colistin tolerance in A. baumannii and underscore the importance of outer membrane structure in both antibiotic resistance and the pathogenesis of A. baumannii.

Global transcriptome analysis of Staphylococcus aureus biofilms in response to innate immune cells.

The potent phagocytic and microbicidal activities of neutrophils and macrophages are among the first lines of defense against bacterial infections. Yet Staphylococcus aureus is often resistant to innate immune defense mechanisms, especially when organized as a biofilm. To investigate how S. aureus biofilms respond to macrophages and neutrophils, gene expression patterns were profiled using Affymetrix microarrays. The addition of macrophages to S. aureus static biofilms led to a global suppression of the biofilm transcriptome with a wide variety of genes downregulated. Notably, genes involved in metabolism, cell wall synthesis/structure, and transcription/translation/replication were among the most highly downregulated, which was most dramatic at 1 h compared to 24 h following macrophage addition to biofilms. Unexpectedly, few genes were enhanced in biofilms after macrophage challenge. Unlike coculture with macrophages, coculture of S. aureus static biofilms with neutrophils did not greatly influence the biofilm transcriptome. Collectively, these experiments demonstrate that S. aureus biofilms differentially modify their gene expression patterns depending on the leukocyte subset encountered.

Diverse genetic regulon of the virulence-associated transcriptional regulator MucR in Brucella abortus 2308.

The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants.

Identification of two small regulatory RNAs linked to virulence in Brucella abortus 2308.

Hfq is an RNA-binding protein that functions in post-transcriptional gene regulation by mediating interactions between mRNAs and small regulatory RNAs (sRNAs). Two proteins encoded by BAB1_1794 and BAB2_0612 are highly over-produced in a Brucella abortus hfq mutant compared with the parental strain, and recently, expression of orthologues of these proteins in Agrobacterium tumefaciens was shown to be regulated by two sRNAs, called AbcR1 and AbcR2. Orthologous sRNAs (likewise designated AbcR1 and AbcR2) have been identified in B. abortus 2308. In Brucella, abcR1 and abcR2 single mutants are not defective in their ability to survive in cultured murine macrophages, but an abcR1 abcR2 double mutant exhibits significant attenuation in macrophages. Additionally, the abcR1 abcR2 double mutant displays significant attenuation in a mouse model of chronic Brucella infection. Quantitative proteomics and microarray analyses revealed that the AbcR sRNAs predominantly regulate genes predicted to be involved in amino acid and polyamine transport and metabolism, and Northern blot analyses indicate that the AbcR sRNAs accelerate the degradation of the target mRNAs. In an Escherichia coli two-plasmid reporter system, overexpression of either AbcR1 or AbcR2 was sufficient for regulation of target mRNAs, indicating that the AbcR sRNAs from B. abortus 2308 perform redundant regulatory functions.

Small molecule inhibitors of Staphylococcus aureus RnpA alter cellular mRNA turnover, exhibit antimicrobial activity, and attenuate pathogenesis.

Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy.

Removal of clinically relevant SARS-CoV-2 variants by an affinity resin containing Galanthus nivalis agglutinin.

The Coronavirus -19 (COVID-19) pandemic due to the SARS-CoV-2 virus has now exceeded two years in duration. The pandemic has been characterized by the development of a succession of variants containing mutations in the spike protein affecting infectiousness, virulence and efficacy of vaccines and monoclonal antibodies. Resistance to vaccination and limitations in the current treatments available require the ongoing development of therapies especially for those with severe disease. The plant lectin Galanthus nivalis binds to mannose structures in the viral envelope. We hypothesized that viral binding should be unaffected by spike protein mutations. Known concentrations of seven clinically relevant SARS-CoV-2 variants were spiked in medium and passed three times over columns containing 1 gm of GNA affinity resin. Percent decrease in viral titer was compared with a control sample. Viral capture efficiency was found to range from 53 to 89% for all variants. Extrapolation indicated that an adult Aethlon Hemopurifier® would have more than sufficient binding capacity for viral loads observed in adult patients with severe COVID-19 infection.

Characterization of components of the Staphylococcus aureus mRNA degradosome holoenzyme-like complex.

Bacterial two-hybrid analysis identified the Staphylococcus aureus RNA degradosome-like complex to include RNase J1, RNase J2, RNase Y, polynucleotide phosphorylase (PNPase), enolase, phosphofructokinase, and a DEAD box RNA helicase. Results also revealed that the recently recognized RNase RnpA interacts with the S. aureus degradosome and that this interaction is conserved in other Gram-positive organisms.

Rsp inhibits attachment and biofilm formation by repressing fnbA in Staphylococcus aureus MW2.

Biofilms contribute to virulence of Staphylococcus aureus. Formation of biofilms is multifactorial, involving polysaccharide, protein, and DNA components, which are controlled by various regulators. Here we report that deletion of the rsp gene resulted in an increase in biofilm formation in strain MW2, suggesting that Rsp is a repressor of biofilm formation. Using SDS-PAGE, we found that Rsp profoundly affected cell surface and secreted proteins. The rsp gene was transcribed monocistronically, and the transcripts were most abundant at the exponential growth phase. Microarray analyses revealed that Rsp represses 75 genes, including 9 genes encoding cell wall-anchored proteins, and activates 22 genes, including 5 genes encoding secreted proteases. Among these genes, fnbA, fnbB, sasG, and spa (which encode cell wall-anchored proteins) and splABCD (which encode secreted proteases) have been implicated in biofilm formation. To deconvolute Rsp's contribution to biofilm formation, we analyzed deletion mutants of these genes either in the wild-type or in the rsp mutant background. We found that fnbA deletion in the rsp mutant restored biofilm formation to the wild-type level, indicating that FnbA plays a major role in Rsp regulation of biofilm formation. Further studies revealed that Rsp inhibited biofilm formation at the stage of primary attachment through repressing fnbA. Rsp belongs to the AraC/XylS family of regulatory proteins. We expressed the putative Rsp DNA binding domain (RspDBD) in Escherichia coli and showed that RspDBD was able to specifically bind to a short DNA fragment containing the fnbA promoter, suggesting that Rsp represses fnbA expression by direct DNA binding.

Both hemolytic anemia and malaria parasite-specific factors increase susceptibility to Nontyphoidal Salmonella enterica serovar typhimurium infection in mice.

Severe pediatric malaria is an important risk factor for developing disseminated infections with nontyphoidal Salmonella serotypes (NTS). While recent animal studies on this subject are lacking, early work suggests that an increased risk for developing systemic NTS infection during malaria is caused by hemolytic anemia, which leads to reduced macrophage microbicidal activity. Here we established a model for oral Salmonella enterica serotype Typhimurium challenge in mice infected with Plasmodium yoelii nigeriensis. Initial characterization of this model showed that 5 days after coinoculation, P. yoelii nigeriensis infection increased the recovery of S. Typhimurium from liver and spleen by approximately 1,000-fold. The increased bacterial burden could be only partially recapitulated by antibody-mediated hemolysis, which increased the recovery of S. Typhimurium from liver and spleen by 10-fold. These data suggested that both hemolysis and P. yoelii nigeriensis-specific factors contributed to the increased susceptibility to S. Typhimurium. The mechanism by which hemolysis impaired resistance to S. Typhimurium was further investigated. In vitro, S. Typhimurium was recovered 24 h after infection of hemophagocytic macrophages in 2-fold-higher numbers than after infection of mock-treated macrophages, making it unlikely that reduced macrophage microbicidal activity was solely responsible for hemolysis-induced immunosuppression during malaria. Infection with P. yoelii nigeriensis, but not antibody-mediated hemolysis, reduced serum levels of interleukin-12p70 (IL-12p70) in response to S. Typhimurium challenge. Collectively, studies establishing a mouse model for this coinfection suggest that multiple distinct malaria-induced immune defects contribute to increased susceptibility to S. Typhimurium.

Establishment of systemic Brucella melitensis infection through the digestive tract requires urease, the type IV secretion system, and lipopolysaccharide O antigen.

Human brucellosis is caused mainly by Brucella melitensis, which is often acquired by ingesting contaminated goat or sheep milk and cheese. Bacterial factors required for food-borne infection of humans by B. melitensis are poorly understood. In this study, a mouse model of oral infection was characterized to assess the roles of urease, the VirB type IV secretion system, and lipopolysaccharide for establishing infection through the digestive tract. B. melitensis strain 16M was consistently recovered from the mesenteric lymph node (MLN), spleen, and liver beginning at 3 or 7 day postinfection (dpi). In the gut, persistence of the inoculum was observed up to 21 dpi. No inflammatory lesions were observed in the ileum or colon during infection. Mutant strains lacking the ureABC genes of the ure1 operon, virB2, or pmm encoding phosphomannomutase were constructed and compared to the wild-type strain for infectivity through the digestive tract. Mutants lacking the virB2 and pmm genes were attenuated in the spleen (P < 0.05) and MLN (P < 0.001), respectively. The wild-type and mutant strains had similar levels of resistance to low pH and 5 or 10% bile, suggesting that the reduced colonization of mutants was not the result of reduced resistance to acid pH or bile salts. In an in vitro lymphoepithelial cell (M-cell) model, B. melitensis transited rapidly through polarized enterocyte monolayers containing M-like cells; however, transit through monolayers containing only enterocytes was reduced or absent. These results indicate that B. melitensis is able to spread systemically from the digestive tract after infection, most likely through M cells of the mucosa-associated lymphoid tissue.

Modulation of the bovine trophoblastic innate immune response by Brucella abortus.

Brucellosis is still a widespread zoonotic disease. Very little is known about the interaction between Brucella abortus and trophoblastic cells, which is essential for better understanding the pathogenesis of the Brucella-induced placentitis and abortion, a key event for transmission of the disease. The goal of this study was to evaluate the profile of gene expression by bovine trophoblastic cells during infection with B. abortus. Explants of chorioallantoic membranes were inoculated with B. abortus strain 2308. Microarray analysis was performed at 4 h after infection, and expression of cytokines and chemokines by trophoblastic cells was assessed by real-time reverse transcription-PCR at 6 and 12 h after inoculation. In addition, cytokine and chemokine expression in placentomes from experimentally infected cows was evaluated. Expression of proinflammatory genes by trophoblastic cells was suppressed at 4 h after inoculation, whereas a significant upregulation of CXC chemokines, namely, CXCL6 (GCP-2) and CXCL8 (interleukin 8), was observed at 12 but not at 6 h after inoculation. Placentomes of experimentally infected cows had a similar profile of chemokine expression, with upregulation of CXCL6 and CXCL8. Our data indicate that B. abortus modulates the innate immune response by trophoblastic cells, suppressing the expression of proinflammatory mediators during the early stages of infection that is followed by a delayed and mild expression of proinflammatory chemokines, which is similar to the profile of chemokine expression in the placentomes of experimentally infected cows. This trophoblastic response is likely to contribute to the pathogenesis of B. abortus-induced placentitis.

The Staphylococcus aureus SrrAB two-component system promotes resistance to nitrosative stress and hypoxia.

UNLABELLED: Staphylococcus aureus is both a commensal and a pathogen of the human host. Survival in the host environment requires resistance to host-derived nitric oxide (NO·). However, S. aureus lacks the NO·-sensing transcriptional regulator NsrR that is used by many bacteria to sense and respond to NO·. In this study, we show that S. aureus is able to sense and respond to both NO· and hypoxia by means of the SrrAB two-component system (TCS). Analysis of the S. aureus transcriptome during nitrosative stress demonstrates the expression of SrrAB-dependent genes required for cytochrome biosynthesis and assembly (qoxABCD, cydAB, hemABCX), anaerobic metabolism (pflAB, adhE, nrdDG), iron-sulfur cluster repair (scdA), and NO· detoxification (hmp). Targeted mutations in SrrAB-regulated loci show that hmp and qoxABCD are required for NO· resistance, whereas nrdDG is specifically required for anaerobic growth. We also show that SrrAB is required for survival in static biofilms, most likely due to oxygen limitation. Activation by hypoxia, NO·, or a qoxABCD quinol oxidase mutation suggests that the SrrAB TCS senses impaired electron flow in the electron transport chain rather than directly interacting with NO· in the manner of NsrR. Nevertheless, like NsrR, SrrAB achieves the physiological goals of selectively expressing hmp in the presence of NO· and minimizing the potential for Fenton chemistry. Activation of the SrrAB regulon allows S. aureus to maintain energy production and essential biosynthetic processes, repair damage, and detoxify NO· in diverse host environments. IMPORTANCE: The Hmp flavohemoglobin is required for nitric oxide resistance and is widely distributed in bacteria. Hmp expression must be tightly regulated, because expression under aerobic conditions in the absence of nitric oxide can exacerbate oxidative stress. In most organisms, hmp expression is controlled by the Fe-S cluster-containing repressor NsrR, but this transcriptional regulator is absent in the human pathogen Staphylococcus aureus. We show here that S. aureus achieves hmp regulation in response to nitric oxide and oxygen limitation by placing it under the control of the SrrAB two-component system, which senses reduced electron flow through the respiratory chain. This provides a striking example of convergent evolution, in which the common physiological goals of responding to nitrosative stress while minimizing Fenton chemistry are achieved by distinct regulatory mechanisms.

Transcriptional profiling of Staphylococcus aureus during growth in 2 M NaCl leads to clarification of physiological roles for Kdp and Ktr K+ uptake systems.

UNLABELLED: Staphylococcus aureus exhibits an unusually high level of osmotolerance and Na(+) tolerance, properties that support survival in various host niches and in preserved foods. The genetic basis of these traits is not well understood. We compared the transcriptional profiles of S. aureus grown in complex medium with and without 2 M NaCl. The stimulon for growth in high-osmolality media and Na(+) included genes involved in uptake of K(+), other compatible solutes, sialic acid, and sugars; capsule biosynthesis; and amino acid and central metabolism. Quantitative PCR analysis revealed that the loci responded differently from each other to high osmolality imposed by elevated NaCl versus sucrose. High-affinity K(+) uptake (kdp) genes and capsule biosynthesis (cap5) genes required the two-component system KdpDE for full induction by osmotic stress, with kdpA induced more by NaCl and cap5B induced more by sucrose. Focusing on K(+) importers, we identified three S. aureus genes belonging to the lower-affinity Trk/Ktr family that encode two membrane proteins (KtrB and KtrD) and one accessory protein (KtrC). In the absence of osmotic stress, the ktr gene transcripts were much more abundant than the kdpA transcript. Disruption of S. aureus kdpA caused a growth defect under low-K(+) conditions, disruption of ktrC resulted in a significant defect in 2 M NaCl, and a ΔktrC ΔkdpA double mutant exhibited both phenotypes. Protective effects of S. aureus Ktr transporters at elevated NaCl are consistent with previous indications that both Na(+) and osmolality challenges are mitigated by the maintenance of a high cytoplasmic K(+) concentration. IMPORTANCE: There is general agreement that the osmotolerance and Na(+) tolerance of Staphylococcus aureus are unusually high for a nonhalophile and support its capacity for human colonization, pathogenesis, and growth in food. Nonetheless, the molecular basis for these properties is not well defined. The genome-wide response of S. aureus to a high concentration, 2 M, of NaCl revealed the upregulation of expected genes, such as those for transporters of compatible solutes that are widely implicated in supporting osmotolerance. A high-affinity potassium uptake system, KdpFABC, was upregulated, although it generally plays a physiological role under very low K(+) conditions. At higher K(+) concentrations, a lower-affinity and more highly expressed type of K(+) transporter system, Ktr transporters, was shown to play a significant role in high Na(+) tolerance. This study illustrates the importance of the K(+) status of the cell for tolerance of Na(+) by S. aureus and underscores the importance of monovalent cation cycles in this pathogen.

PPARγ-mediated increase in glucose availability sustains chronic Brucella abortus infection in alternatively activated macrophages.

Eradication of persistent intracellular bacterial pathogens with antibiotic therapy is often slow or incomplete. However, strategies to augment antibiotics are hampered by our poor understanding of the nutritional environment that sustains chronic infection. Here we show that the intracellular pathogen Brucella abortus survives and replicates preferentially in alternatively activated macrophages (AAMs), which are more abundant during chronic infection. A metabolic shift induced by peroxisome proliferator-activated receptor γ (PPARγ), which increases intracellular glucose availability, is identified as a causal mechanism promoting enhanced bacterial survival in AAMs. Glucose uptake was crucial for increased replication of B. abortus in AAMs, and for chronic infection, as inactivation of the bacterial glucose transporter gluP reduced both intracellular survival in AAMs and persistence in mice. Thus, a shift in intracellular nutrient availability induced by PPARγ promotes chronic persistence of B. abortus within AAMs, and targeting this pathway may aid in eradicating chronic infection.

RNA decay: a novel therapeutic target in bacteria.

The need for novel antibiotics is greater now than perhaps any time since the pre-antibiotic era. Indeed, the recent collapse of most pharmaceutical antibacterial groups, combined with the emergence of hypervirulent and pan-antibiotic-resistant bacteria have, in effect, created a 'perfect storm' that has severely compromised infection treatment options and led to dramatic increases in the incidence and severity of bacterial infections. To put simply, it is imperative that we develop new classes of antibiotics for the therapeutic intervention of bacterial infections. In that regard, RNA degradation is an essential biological process that has not been exploited for antibiotic development. Herein we discuss the factors that govern bacterial RNA degradation, highlight members of this machinery that represent attractive antimicrobial drug development targets and describe the use of high-throughput screening as a means of developing antimicrobials that target these enzymes. Such agents would represent first-in-class antibiotics that would be less apt to inactivation by currently encountered enzymatic antibiotic-resistance determinants.

The small protein CydX is required for function of cytochrome bd oxidase in Brucella abortus.

A large number of hypothetical genes potentially encoding small proteins of unknown function are annotated in the Brucella abortus genome. Individual deletion of 30 of these genes identified four mutants, in BAB1_0355, BAB2_0726, BAB2_0470, and BAB2_0450 that were highly attenuated for infection. BAB2_0726, an YbgT-family protein located at the 3' end of the cydAB genes encoding cytochrome bd ubiquinal oxidase, was designated cydX. A B. abortus cydX mutant lacked cytochrome bd oxidase activity, as shown by increased sensitivity to H(2)O(2), decreased acid tolerance and increased resistance to killing by respiratory inhibitors. The C terminus, but not the N terminus, of CydX was located in the periplasm, suggesting that CydX is an integral cytoplasmic membrane protein. Phenotypic analysis of the cydX mutant, therefore, suggested that CydX is required for full function of cytochrome bd oxidase, possibly via regulation of its assembly or activity.

Staphylococcus aureus nonribosomal peptide secondary metabolites regulate virulence.

Staphylococcus aureus is a major human pathogen that is resistant to numerous antibiotics in clinical use. We found two nonribosomal peptide secondary metabolites--the aureusimines, made by S. aureus--that are not antibiotics, but function as regulators of virulence factor expression and are necessary for productive infections. In vivo mouse models of bacteremia showed that strains of S. aureus unable to produce aureusimines were attenuated and/or cleared from major organs, including the spleen, liver, and heart. Targeting aureusimine synthesis may offer novel leads for anti-infective drugs.