Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction

Acinetobacter baumannii OxyR regulates the transcriptional response to hydrogen peroxide

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes diverse infections, including pneumonia, bacteremia, and wound infections. Due to multiple intrinsic and acquired antimicrobial-resistance mechanisms, A. baumannii isolates are commonly multidrug resistant, and infections are notoriously difficult to treat. The World Health Organization recently highlighted carbapenem-resistant A. baumannii as a "critical priority" for the development of new antimicrobials because of the risk to human health posed by this organism. Therefore, it is important to discover the mechanisms used by A. baumannii to survive stresses encountered during infection in order to identify new drug targets. In this study, by use of in vivo imaging, we identified hydrogen peroxide (H2O2) as a stressor produced in the lung during A. baumannii infection and defined OxyR as a transcriptional regulator of the H2O2 stress response. Upon exposure to H2O2, A. baumannii differentially transcribes several hundred genes. However, the transcriptional upregulation of genes predicted to detoxify hydrogen peroxide is abolished in an A. baumannii strain in which the transcriptional regulator oxyR is genetically inactivated. Moreover, inactivation of oxyR in both antimicrobial-susceptible and multidrug-resistant A. baumannii strains impairs growth in the presence of H2O2 OxyR is a direct regulator of katE and ahpF1, which encode the major H2O2-degrading enzymes in A. baumannii, as confirmed through measurement of promoter binding by recombinant OxyR in electromobility shift assays. Finally, an oxyR mutant is less fit than wild-type A. baumannii during infection of the murine lung. This work reveals a mechanism used by this important human pathogen to survive H2O2 stress encountered during infection

Genetic variation effects growth inhibition by glyphosate.

<p><b>A</b>. Shikimate pathway produces the precursor for phenylalanine, tyrosine, tryptophan, <i>para</i>-Aminobenzoic acid (PABA), folic acid and Coenzyme Q10. The canonical target of glyphosate is EPSPS in plants and Aro1 is the yeast homolog of EPSPS. PABA and 4-hydroxylbenzoate can be converted to Coenzyme Q10. <b>B</b>. Serial dilution of genetically diverse yeast on rich media (YPD) with dilutions of glyphosate as indicated (1% vol/ vol is equivalent to 78 mM). <b>C</b>. Serial dilution of genetically diverse yeast on minimal media with glyphosate. Aromatic amino acids, tryptophan (W), tyrosine (Y) and phenylalanine (F) were added to YM plates to make WYF.</p

In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection.

Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe2+ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe2+ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology

Genetic linkage analysis of glyphosate sensitivity in glyphosate on rich media.

<p><b>A</b>. Genetic linkage of sensitivity of YJM789 to 1% glyphosate in YPD. LOD score (y-axis) was mapped across the yeast genome (x-axis) with chromosomes numbered left to right. The LOD significant levels (alpha = 0.05) was 3.82 and was marked by a red dashed line. <b>B</b>. The genomic loci under the peak located on chromosome 15 contains 10 genes. Genes encoded on the top strand are in yellow and genes encoded on the bottom strand are blue. <b>C</b>. Serial dilution of S288c (GSY147) x YJM789 hybrids in which the entire <i>PDR5</i> coding region in either parent was deleted (Δ), crossed, and the resulting hemizygotes were grown on rich media with and without 1% of glyphosate.</p

Regulation of <i>DIP5</i> by glyphosate and aspartic acid.

<p><b>A</b>. RNA expression levels of <i>DIP5</i> mRNA in AWRI1631, RM11, YJM789 and S288c grown in YM with and without 0.25% glyphosate (RU). Q RT-PCR of <i>DIP5</i> mRNA levels are normalized to <i>ACT1</i> mRNA. <b>B</b>. Serial dilution of genetically diverse yeast on minimal media with glyphosate supplemented with aspartic acid. <b>C</b>. Different responses of <i>dip5Δ</i> yeast in liquid media supplemented with different aromatic metabolites and amino acids on exposure to glyphosate. BY4741 and GSY147 were grown in the presence of glyphosate (0.1% in HULM, WYF and D and 1% in YPD) and the optical density was measured in log phase (10 hr).</p

Genetic variation effects growth inhibition by glyphosate.

<p><b>A</b>. Shikimate pathway produces the precursor for phenylalanine, tyrosine, tryptophan, <i>para</i>-Aminobenzoic acid (PABA), folic acid and Coenzyme Q10. The canonical target of glyphosate is EPSPS in plants and Aro1 is the yeast homolog of EPSPS. PABA and 4-hydroxylbenzoate can be converted to Coenzyme Q10. <b>B</b>. Serial dilution of genetically diverse yeast on rich media (YPD) with dilutions of glyphosate as indicated (1% vol/ vol is equivalent to 78 mM). <b>C</b>. Serial dilution of genetically diverse yeast on minimal media with glyphosate. Aromatic amino acids, tryptophan (W), tyrosine (Y) and phenylalanine (F) were added to YM plates to make WYF.</p

Genetic Linkage analysis of glyphosate sensitivity in glyphosate on minimal media with and without aromatic amino acids.

<p><b>A</b>. Genetic linkage of sensitivity of YJM789 to 0.15% glyphosate in yeast minimal media supplemented with aromatic amino acids (WYF). LOD score (y-axis) was mapped across the yeast genome (x-axis). The LOD significant levels (alpha = 0.05) was 3.65 and was marked by a red dashed line. <b>B</b>. Genetic linkage of sensitivity of YJM789 to 0.15% glyphosate in yeast minimal media (YM). LOD score (y-axis) was mapped across the yeast genome (x-axis). The LOD significant levels (alpha = 0.05) was 3.47 and was marked by a red dashed line. <b>C</b>. The genomic loci under the peak located on chromosome 16 contains 14 genes. Genes encoded on the top strand are in yellow and genes encoded on the bottom strand are blue. <b>D</b>. Serial dilutions of S288c (GSY147), YJM789 and BY4741 with <i>DIP5</i> knocked out grown on YM (HULM), WYF, aspartic acid (D) with glyphosate at the concentrations indicated. Histidine, uracil, leucine and methionine (HULM) were supplemented for growth of BY4741. <b>E</b>. Serial dilutions of S288c (GSY147) x YJM789 hybrids. The entire <i>DIP5</i> coding region in either parent was deleted (Δ), crossed, and the resulting hemizygotes were grown on solid media YM, WYF, aspartic acid (D) with glyphosate at the indicated concentrations and supplemented.</p

Contribution of the genetic variation within Aro1 to glyphosate resistance.

<p><b>A</b>. Protein alignment of Aro1 from genetically diverse yeast strains. <b>B</b>. Serial dilutions of haploid <i>aro1Δ</i> in RM11, <b>C</b>. S288c, and <b>D</b>. YJM789, expressing different alleles of plasmid encoded <i>ARO1</i> were grown on YPD and YM with and without glyphosate. Rows labeled empty have pGS36 plasmid with no <i>ARO1</i>. Parental strains with the endogenous <i>ARO1</i> expressed from the chromosome labeled WT carry an empty plasmid (pGS36).</p

Genetic variation in Dip5, an amino acid permease, and Pdr5, a multiple drug transporter, regulates glyphosate resistance in <i>S</i>. <i>cerevisiae</i> - Fig 3

<p><b>A. Different responses of <i>aro1</i></b><i>Δ</i><b>yeast on media supplemented with different aromatic metabolites</b>. Wild-type RM11 was compared to S288c (GSY147) and YJM789 with and without <i>ARO1</i>, three days in minimal media supplemented with aromatic amino acids (+WYF) or <i>para</i>-aminobenzoic acid (+PABA) or without these metabolites (-WYF or -PABA). <b>B</b>. Different responses of <i>aro1Δ</i> yeast on glyphosate. Wild-type RM11 was compared to S288c (GSY147) and YJM789 with and without <i>ARO1</i> on 0.15% glyphosate three days in minimal media supplemented with +WYF or +PABA or without these metabolites (-WYF or -PABA).</p