Functional Importance of Proline Transporters in Staphylococcus aureus

Staphylococcus aureus can survive and colonize a multitude of environmental niches including the human host. S. aureus is responsible for over 11,000 deaths yearly, and many studies correlate the survival and proliferation of S. aureus to its acquisition and utilization of proline. Proline is an important amino acid as it serves as both an osmotic protectant and carbon source. It was determined that S. aureus had a severe growth defect when grown in osmotically stressed conditions without proline. Additionally, in an abscess environment, where it is hypothesized that the primary carbon sources (i.e. glucose) are depleted, S. aureus will acquire proline from the extracellular milieu as a secondary carbon source. The transport of proline occurs through three transporters, a high-affinity proline permease, PutP and two low-affinity proline transporters, OpuD and ProP. In this study, we sought to investigate the function and regulation of the three transporters. It was determined that CcpA negatively regulated the expression of the three transporters, while σB (SigB) negatively regulated the expression of putP. Finally, we found that osmotic stress upregulates the expression of opuD and proP, but downregulates putP expression

Loss of CFTR function in macrophages alters the cell transcriptional program and delays lung resolution of inflammation

Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the CF Transmembrane-conductance Regulator (CFTR) gene. The most severe pathologies of CF occur in the lung, manifesting as chronic bacterial infection, persistent neutrophilic inflammation, and mucopurulent airway obstruction. Despite increasing knowledge of the CF primary defect and the resulting clinical sequelae, the relationship between the CFTR loss of function and the neutrophilic inflammation remains incompletely understood. Here, we report that loss of CFTR function in macrophages causes extended lung inflammation. After intratracheal inoculation with Pseudomonas aeruginosa, mice with a macrophage-specific Cftr-knockout (Mac-CF) were able to mount an effective host defense to clear the bacterial infection. However, three days post-inoculation, Mac-CF lungs demonstrated significantly more neutrophil infiltration and higher levels of inflammatory cytokines, suggesting that Mac-CF mice had a slower resolution of inflammation. Single-cell RNA sequencing revealed that absence of CFTR in the macrophages altered the cell transcriptional program, affecting the cell inflammatory and immune responses, antioxidant system, and mitochondrial respiration. Thus, loss of CFTR function in macrophages influences cell homeostasis, leading to a dysregulated cellular response to infection that may exacerbate CF lung disease

Myeloid CFTR loss‐of‐function causes persistent neutrophilic inflammation in cystic fibrosis

Persistent neutrophilic inflammation is a hallmark of cystic fibrosis (CF). However, the mechanisms underlying this outstanding pathology remain incompletely understood. Here, we report that CFTR in myeloid immune cells plays a pivotal role in control of neutrophilic inflammation. Myeloid CFTR‐Knockout (Mye‐Cftr−/−) mice and congenic wild‐type (WT) mice were challenged peritoneally with zymosan particles at different doses, creating aseptic peritonitis with varied severity. A high‐dose challenge resulted in significantly higher mortality in Mye‐Cftr−/− mice, indicating an intrinsic defect in host control of inflammation in mice whose myeloid cells lack CF. The low‐dose challenge demonstrated an impaired resolution of inflammation in Mye‐Cftr−/− mice, reflected by a significant overproduction of proinflammatory cytokines, including neutrophil chemokines MIP‐2 and KC, and sustained accumulation of neutrophils. Tracing neutrophil mobilization in vivo demonstrated that myeloid CF mice recruited significantly more neutrophils than did WT mice. Pulmonary challenge with zymosan elicited exuberant inflammation in the lung and recapitulated the findings from peritoneal challenge. To determine the major type of cell that was primarily responsible for the over‐recruitment of neutrophils, we purified and cultured ex vivo zymosan‐elicited peritoneal neutrophils and macrophages. The CF neutrophils produced significantly more MIP‐2 than did the WT counterparts, and peripheral blood neutrophils isolated from myeloid CF mice also produced significantly more MIP‐2 after zymosan stimulation in vitro. These data altogether suggest that CFTR dysfunction in myeloid immune cells, especially neutrophils, leads to hyperinflammation and excessive neutrophil mobilization in the absence of infection. Thus, dysregulated inflammation secondary to abnormal or absent CFTR in myeloid cells may underlie the clinically observed neutrophilic inflammation in CF.Graphical AbstractMyeloid CFTR dysfunction underlies the clinically observed neutrophilic inflammation in cystic fibrosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163561/2/jlb10687_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163561/1/jlb10687.pd

ProT is highly conserved among staphylococcal species.

ProT orthologues from representative staphylococcal species were aligned using Jalview 2.11.2.4. The referenced alleles include: ARG45194.1 (S. aureus), WP_207517961.1 (S. simiae), WP_064212334.1 (S. capitis), WP_251481646.1 (S. xylosus), WP_087436598.1 (S. hominis), WP_107614115.1 (S. haemolyticus), WP_150873555.1 (S. saprophyticus), and WP_107511203.1 (S. epidermidis). (TIF)</p

Proline is not transported by OpuC, OpuD, or ProP.

A) 3H-proline transport assays, B) measured amounts of proline accumulated at 10 minutes, and C) growth analysis in CDM and CDM-P of JE2 and penta pBK123 (empty vector), pML1 (Pcad::proT), pML2 (Pcad::opuC), pML3 (Pcad::opuD), pML4 (Pcad::proP), and pML5 (Pcad::putP) confirm OpuC, OpuD, and ProP do not transport proline under these conditions. 3H-proline transport data (A) are standardized to JE2 to account for experimental variations and are fit by nonlinear regression using a first-order equation of technical duplicates ran three independent times. Accumulated proline data (B) are represented by the mean ± SD (n = 5–6). Kruskal-Wallis multiple comparisons identified statistically significant differences (* p<0.05). Growth analysis data (C) are represented by the mean ± SD (n = 3).</p

Primers used in these studies.

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Proline transport is required for organ colonization in a bacteremia model.

Bacterial burdens of 7-week-old C57BL/6 mice retro-orbitally inoculated with 5x106 of S. aureus JE2, penta mutant, ΔproT ΔputP, Δ4-proT, and Δ4-putP 5 days post-infection in the kidneys and liver were determined. Data are represented by the median with statistical significance determined by Mann-Whitney test. p values are defined on the graph. (TIF)</p

ProT is the primary proline transporter in the presence of high salt.

Growth analysis of the following strains in CDM supplemented with 1 M NaCl: A) JE2, penta, Δ4-proT, Δ4-opuC, Δ4-opuD, Δ4-proP, and Δ4-putP B) JE2, penta, ΔproT, ΔopuC, ΔopuD, ΔproP, and ΔputP C) JE2, ΔproC, ΔproT, and ΔproT ΔproC D) JE2 empty vector, penta empty vector, penta Pcad::proT, penta Pcad::opuC, penta Pcad::opuD, penta Pcad::proP, and penta Pcad::putP reveal ProT is important for maximal growth under these conditions. Data are represented by the mean ± SD (n = 2–3).</p