Utilizing Understudied Bacteria in the Pursuit of Natural Products Drug Discovery

For decades natural products have been a leading source of bioactive compounds that have had a profound impact on human health through their development into clinical pharmaceuticals. However, the private sector has moved away from this pursuit. This divestment of microbial drug discovery can be attributed to several factors, the most common being: i) continued rediscovery of known chemical entities, and ii) a move towards small molecules generated through combinatorial chemistry and high throughput screening (HTS). Nonetheless, academic researchers continued to see the promise of bacterial secondary metabolites with advances in genomics and metabolomics, suggesting that the microbial world still encodes for a reservoir of yet described chemical entities. We opine that the field must move towards the direction of targeting understudied bacterial phyla or understudied bacterial genera within well investigated phyla to access this new chemical space. Herein we describe a novel mass spectrometry pipeline to achieve this. We assert that using MALDI MS and IDBac in the front end of discovery programs will afford researchers an inexpensive, rapid, and precise method to mine large environmental collections of isolates for understudied or target bacteria. While then investigating an understudied bacterial isolate Chitinophaga AC21 to demonstrate the promise that understudied bacteria show for natural products drug discovery

Effect of burn injury and wound infection with or without IL-15 SA treatment on CD4⁺, CD8⁺ T and CD19⁺ B lymphocytes activation status.

<p>CD69 surface expression was used as a marker of cellular activation and measured by flow cytometry. Upper half of the panel depicts data for spleen cells and lower half of the panel depicts data for liver tissue. Percentage of CD69 expression was measured on CD4⁺ T cells <b>(A</b> and <b>D)</b>, CD8⁺ T cells <b>(B</b> and <b>E</b>), and CD3^-CD19⁺ B cells <b>(C</b> and <b>F</b>); in the spleen and liver. n = 8–12 in each group and P<0.05. *significantly different as compared to respective day 4 and day 6 sham groups; #significantly different from day 4 burn injury group (B+V), and $—significantly different from day 6 burn injury + wound infection + vehicle treated group (B+WI+V).</p

Burn wound infection caused decline in white blood cell and lymphocyte counts.

<p><b>(A)</b> Total white blood cell counts. <b>(B)</b> Blood lymphocyte counts. (C) Blood neutrophil counts. n = 8–10 in each group, *significantly different as compared to respective day 4 and day 6 sham groups; #significantly different from burn plus vehicle group (B+V) and P<0.05.</p

IL-15 SA treatment elicits expansion of NK and NKT cells post burn injury and wound infection causes their decline.

<p>Upper half and lower half of the panel depicts cell counts and activation status (CD69⁺) of NK (CD3^-CD49b⁺) and NKT (CD3⁺CD49b^-) cells in the spleen and liver respectively. The graphs <b>A</b> and <b>E</b> depict NK cell counts in the spleen and liver respectively; and the graphs <b>B</b> and <b>F</b> show the NKT cell counts in the spleen and liver respectively. The graphs <b>C</b> and <b>G</b> depict NK cell CD69 expression in the spleen and liver respectively; and the graph <b>D</b> shows the NKT cell CD69 expression in the spleen. n = 8–10 in each group and P<0.05. *significantly different as compared to respective day 4 and day 6 sham groups; #significantly different from day 4 burn injury group (B+V), and $—significantly different from day 6 burn injury + wound infection + vehicle treated group (B+WI+V).</p

Effect of burn injury and wound infection with or without IL-15 SA treatment on CD4⁺, CD8⁺ T and CD19⁺ B lymphocyte cell counts.

<p>Wound infection and sepsis was induced by inoculation of <i>Pseudomonas aeruginosa</i> on day 4 post burn injury. Upper half of the panel depicts cell counts in the spleen and lower half of the panel depicts cell counts in the liver. Spleen and liver cell counts were analyzed on day 4, and day 6 (equivalent to day 2 post wound infection) post burn injury. <b>(A</b> and <b>G)</b> Total immune cell counts among the respective groups of the single cell suspensions prepared from the spleen and liver. Flow cytometry was used to measure the individual T and B cell lymphocyte cell population in the spleen and liver. The bar graph represents the total cell counts of the CD4⁺ T cells <b>(B</b> and <b>H)</b>, CD4⁺ CD44^hi memory T cells <b>(D</b> and <b>J</b>), CD8⁺ T cells <b>(C</b> and <b>I</b>), CD8⁺ CD44^hi T cells <b>(E</b> and <b>K)</b>, and CD3^-CD19⁺ B cells <b>(F</b> and <b>L</b>); in the spleen and liver. n = 8–12 in each group and P<0.05. *significantly different as compared to respective day 4 and day 6 sham groups; #significantly different from day 4 burn injury group (B+V) and $-significantly different from day 6 burn injury + vehicle treated wound infected group (B+WI+V).</p

Effect of IL-15 SA treatment on neutrophil recruitment and bacterial clearance upon intraperitoneal infection.

<p><i>Pseudomonas aeruginosa</i> (1x10⁸ CFU) was inoculated in the peritoneum of the mice via i.p injection. This model was used to assess if IL-15 SA treatment causes neutrophil recruitment and improves bacterial clearance. Mice were divided into four groups: 1) <b>B+V</b> = burn + vehicle treated non-infected mice; 2) <b>B+IL-15</b> = burn + IL-15 SA treated non-infected mice; 3) <b>B+V+I</b> = burn + vehicle treated infected mice; 4) <b>B+IL-15+I</b> = burn + IL-15 SA treated infected mice. <b>(A)</b> At 6 hours post i.p infection, core body temperature was measured using a rectal temperature probe. Also, peritoneal lavage was performed using 2 mL sterile PBS solution and used for bacterial clearance <b>(B)</b>; macrophage <b>(C)</b> and neutrophil <b>(D)</b> cell counts measurements using flow cytometry. n = 8–16 in each group and P<0.05. *significantly different as compared to B+V group; #significantly different from B+IL-15 group, significantly different as compared to B+V group $—significantly different from B+V+I group.</p

Plasma cytokine levels post intraperitoneal infection with <i>Pseudomonas aeruginosa.</i>

<p>Plasma cytokines including (A) IL-6, (B) IL-10, (C) TNF-α, (D) IFN-γ, (E) KC and (F) MIP-2 were measured using Bio-Rad Bio-Plex assay. n = 6–8 in each group and P<0.05. *significantly different as compared to burn injury + vehicle treated group (B+V); #significantly different from burn injury + IL-15 SA treated group (B+IL-15); and $significantly different from burn injury + IL-15 SA treated infected group (B+IL-15+I).</p

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