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

Depletion of METTL3 alters cellular and extracellular levels of miRNAs containing m6A consensus sequences

Extracellular vesicles (EVs) are capable of transferring cargo from donor to recipient cells, but precisely how cargo content is regulated for export is mostly unknown. For miRNA cargo, we previously showed that when compared to isogenic colorectal cancer (CRC) cells expressing wild-type KRAS, a distinct subset of miRNAs are differentially enriched in EVs from KRAS mutant active CRC cells, with miR-100 being one of the most enriched. The mechanisms that could explain how miR-100 and other miRNAs are differentially exported into EVs have not been fully elucidated. Here, we tested the effect of N6-methyladenosine (m6A) modification on miRNA export into EVs by depletion of METTL3 and ALKBH5, a writer and eraser of m6A modification, respectively. While the effects of ALKBH5 knockdown were quite modest, decreased levels of METTL3 led to reduced cellular and extracellular levels of a subset of miRNAs that contain consensus sequences for m6A modification. Functional testing of EVs prepared from cells expressing shRNAs against METTL3 showed that they were less capable of conferring colony growth in 3D to wild-type KRAS cells and were also largely incapable of conferring the spread of cetuximab resistance. Our data support a role for METTL3 modification on cellular miRNA levels and export of specific miRNAs

Diverse Long RNAs Are Differentially Sorted into Extracellular Vesicles Secreted by Colorectal Cancer Cells

Summary: The regulation and functional roles of secreted coding and long noncoding RNAs (lncRNAs; >200 nt) are largely unknown. We previously showed that mutant KRAS colorectal cancer (CRC) cells release extracellular vesicles (EVs) containing distinct proteomes, microRNAs (miRNAs), and circular RNAs. Here, we comprehensively identify diverse classes of CRC extracellular long RNAs secreted in EVs and demonstrate differential export of specific RNAs. Distinct noncoding RNAs, including antisense transcripts and transcripts derived from pseudogenes, are enriched in EVs compared to cellular profiles. We detected strong enrichment of Rab13 in mutant KRAS EVs and demonstrate functional delivery of Rab13 mRNA to recipient cells. To assay functional transfer of lncRNAs, we implemented a CRISPR/Cas9-based RNA-tracking system to monitor delivery to recipient cells. We show that gRNAs containing export signals from secreted RNAs can be transferred from donor to recipient cells. Our data support the existence of cellular mechanisms to selectively export diverse classes of RNA. : Extracellular vesicles (EVs) contain protein and RNA cargo that can be transferred between cells. Hinger et al. identify distinct subsets of cellular coding and long noncoding RNAs that are enriched in EVs that can be functionally transferred between cells, supporting a regulated form of cell-cell communication. Keywords: extracellular vesicles, exosomes, lncRNA, Rab13, extracellular RNA, RNAseq, CRISPR display, colorectal cancer, KRA

Neutrophil depletion rescues the intraosseous growth defect of an <i>srrA</i> mutant.

<p>Mice were given serial injections of anti-Ly6G monoclonal antibody. As a control, mice received injections of an isotype control antibody. At 14 days post-infection, femurs were processed for CFU enumeration. N = 4 mice per group. Horizontal lines represent the mean. Error bars represent SD. Significance determined by Students <i>t</i> test.</p

SrrAB is required for intraosseous survival and cortical bone destruction during <i>S</i>. <i>aureus</i> osteomyelitis.

<p>Osteomyelitis was induced in groups of mice using WT or Δ<i>srrA</i> strains. (A) At 5 and 14 days post-infection, femurs were processed for colony forming units (CFU) enumeration. N = 5 mice per group. Horizontal line represents the mean and error bars represent SD. (B and C) Antero-posterior views of WT (B) or Δ<i>srrA</i> (C) infected femurs at 14 days post-inoculation. (D) MicroCT imaging analysis of cortical bone destruction (mm³) 14 days post-inoculation. N = 4 mice per group. Error bars represent the SEM. Statistical significance determined by Students <i>t</i> test.</p

<i>S</i>. <i>aureus</i> modulates quorum sensing and exotoxin production in response to oxygenation.

<p>Supernatants from WT or Δ<i>srrA</i> were prepared by inoculating RPMI and 1% CA with a 1:1000 dilution from overnight cultures and growing for 15 hours either aerobically or hypoxically. Identical culture conditions were used to monitor quorum sensing and transcript levels (see below). (A) MC3T3 cells were seeded into 96 well plates at 5,000 cells per well. After 24 hours, growth media was replaced, and 20% or 30% of the total media volume was replaced with concentrated culture supernatant grown either aerobically or hypoxically, or an equivalent volume of RPMI. Cell viability was assessed 24 hours later, and results are expressed as percent of RPMI control (n = 10). Results are representative of at least three independent experiments. Error bars represent the SEM. (B) Agr-mediated quorum sensing was monitored using <i>agr</i>P3-dependent YFP expression in WT or Δ<i>srrA</i> strains grown aerobically or hypoxically as above. YFP relative fluorescent units (RFUs) were averaged from 3 technical replicates. Error bars represent the SD. Data shown are an average of 3 biologically independent experiments. RFUs monitored at 0, 6, 9, 12, and 15 hours after back-dilution from overnight culture. * and ** represent <i>p</i><0.05 and 0.01, respectively relative to WT aerobic at 15 hours as determined by Student’s <i>t</i> test. # represents <i>p</i><0.05 relative to Δ<i>srrA</i> aerobic. (C) cDNA samples from WT or Δ<i>srrA</i> strains grown aerobically or hypoxically as above for 15 hours were subjected to qRT-PCR. Graph depicts fold change of the indicated transcripts relative to WT aerobic transcript level. Data shown are an average of 3 biologically independent experiments. Error bars represent the SEM. Significance was determined by two way ANOVA. * denotes <i>p</i><0.05, ** denotes <i>p</i><0.01, and *** denotes <i>p</i><0.001 relative to WT aerobic.</p

<i>S</i>. <i>aureus</i> osteomyelitis triggers reduced oxygen availability in skeletal tissues.

<p>Oxygen tension (pO₂) was measured in murine femurs infected with <i>S</i>. <i>aureus</i> (black circles) at 1, 4, 7 and 10 days post-infection (n = 6 from two independent experiments). Uninfected femurs (open squares) were measured for oxygen tension immediately following (n = 5) or 4 days after (n = 3) a mock inoculation procedure. Oxygen tension is reported as mmHg. Horizontal lines represent the mean. Error bars represent the SD. Dotted line represents the upper limit of detection.</p

The <i>srrAB</i> promoter is active in hypoxic skeletal tissues.

<p>Groups of mice (n = 3 per group) were subjected to osteomyelitis by infection with WT bacteria containing either P<i>srrAB</i>-pAmiLux or pAmiLux (promoterless control). At 1 or 24 hours post-inoculation, infected femurs were explanted and immediately imaged on an IVIS 200 system (5 minute exposure).</p