{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T10:46:22Z","timestamp":1765277182033,"version":"3.46.0"},"posted":{"date-parts":[[2025,12,5]]},"group-title":"Microbiology","reference-count":62,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,5]],"date-time":"2025-12-05T00:00:00Z","timestamp":1764892800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,5]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The serine\/threonine protein kinase F (PknF) of\n                  <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                  (Mtb) has poorly defined targets and functions but is involved in limiting NLRP3 inflammasome activation in murine macrophages and dendritic cells\n                  <jats:italic>in vitro<\/jats:italic>\n                  . The importance of PknF for the virulence of Mtb\n                  <jats:italic>in vivo<\/jats:italic>\n                  is not known. Here, we demonstrate that the Mtb CDC1551 deletion mutant of\n                  <jats:italic>pknF (<\/jats:italic>\n                  \u0394\n                  <jats:italic>pknF<\/jats:italic>\n                  ) expresses significantly increased levels of the lipid pthiocerol dimycoserosate (PDIM), a polyketide lipid with pro-virulence properties. The \u0394\n                  <jats:italic>pknF<\/jats:italic>\n                  mutant strain, when compared to the Mtb and complemented strains, had a 100-fold increase in growth at day 28 and about a 10-fold increase in growth at days 90-98 in the lungs of mice. The increase in pulmonary bacterial loads after infection with \u0394\n                  <jats:italic>pknF<\/jats:italic>\n                  strain was conserved even in\n                  <jats:italic>Nlrp3<\/jats:italic>\n                  -deficient mice, arguing that PknF modulates Mtb virulence independently of NLRP3-inflammasome activation in mice. Staining of lung sections revealed increased inflammation in the lungs of \u0394\n                  <jats:italic>pknF<\/jats:italic>\n                  strain-infected mice when compared to Mtb and the complemented mutant strain. Highly susceptible B6.Sst1\n                  <jats:sup>S<\/jats:sup>\n                  mice displayed decreased host resistance with significantly decreased survival when infected with the \u0394\n                  <jats:italic>pknF<\/jats:italic>\n                  strain compared to the complemented strain or Mtb. In conclusion, our data suggest that expression of PknF, as a modulator of multiple downstream effector proteins, restricts the virulence of Mtb in the lungs of mice through an NLRP3 inflammasome-independent mechanism but potentially via suppressing expression of the virulence lipid, PDIM.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Author summary<\/jats:title>\n                  <jats:p>\n                    The human pathogen\n                    <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    (Mtb) encodes 11 serine\/threonine protein kinases (Pkn A-I,K and L).\n                    <jats:italic>In vitro<\/jats:italic>\n                    , PknF inhibits activation of innate immune responses in macrophages and dendritic cells. The importance of that protein for the virulence of the bacteria in the context of a live animal infection is unknown. We used a hypersusceptible mouse strain to analyze the impact of deleting the\n                    <jats:italic>pknF<\/jats:italic>\n                    gene on the virulence of the bacteria by monitoring the survival of the mice. We show that the \u0394\n                    <jats:italic>pknF<\/jats:italic>\n                    deletion mutant kills mice faster than wild-type bacteria or \u0394\n                    <jats:italic>pknF<\/jats:italic>\n                    mutant bacteria expressing a wild-type copy of the\n                    <jats:italic>pknF<\/jats:italic>\n                    gene (\u0394\n                    <jats:italic>pknF<\/jats:italic>\n                    -C). Next, we analyzed the growth of the different bacterial strains in the infected mice and demonstrated that at day 28 and day 90-98 timepoints the \u0394\n                    <jats:italic>pknF<\/jats:italic>\n                    mutant bacterial strains showed a significant increase in bacterial burden in the lungs, bronchoalveolar lavage fluid and the spleen. An analysis of the total lipids of the bacterial stocks showed that the \u0394\n                    <jats:italic>pknF<\/jats:italic>\n                    Mtb bacteria increased expression of the virulence lipid, pthiocerol dimycoserosate (PDIM), suggesting a negative regulation of this lipid by PknF likely affecting bacterial virulence in the lungs of mice.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2025.12.04.692467","type":"posted-content","created":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T12:31:59Z","timestamp":1765197119000},"source":"Crossref","is-referenced-by-count":0,"title":["Protein kinase F regulates the virulence of\n                  <i>Mycobacterium tuberculosis<\/i>"],"prefix":"10.64898","author":[{"given":"Flor","family":"Torres-Juarez","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0046-0045","authenticated-orcid":false,"given":"Shivangi","family":"Rastogi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Young","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul J.","family":"Baker","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Viplov K.","family":"Biswas","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shannon E.","family":"Khan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"D. Branch","family":"Moody","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katrin D.","family":"Mayer-Barber","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5830-6107","authenticated-orcid":false,"given":"Volker","family":"Briken","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025120902301476000_2025.12.04.692467v1.1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1009712"},{"key":"2025120902301476000_2025.12.04.692467v1.2","doi-asserted-by":"crossref","first-page":"690","DOI":"10.4049\/jimmunol.2300753","article-title":"Mycobacterium tuberculosis Utilizes Serine\/Threonine Kinase PknF to Evade NLRP3 Inflammasome-driven Caspase-1 and RIPK3\/Caspase-8 Activation in Murine Dendritic Cells","volume":"213","year":"2024","journal-title":"J Immunol"},{"key":"2025120902301476000_2025.12.04.692467v1.3","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.0904189"},{"key":"2025120902301476000_2025.12.04.692467v1.4","doi-asserted-by":"publisher","DOI":"10.1111\/imr.12249"},{"key":"2025120902301476000_2025.12.04.692467v1.5","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.179.2.1178"},{"key":"2025120902301476000_2025.12.04.692467v1.6","doi-asserted-by":"publisher","DOI":"10.1111\/j.1348-0421.2001.tb01310.x"},{"key":"2025120902301476000_2025.12.04.692467v1.7","doi-asserted-by":"publisher","DOI":"10.1086\/315771"},{"key":"2025120902301476000_2025.12.04.692467v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/labinvest.3780079"},{"key":"2025120902301476000_2025.12.04.692467v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2011.12.002"},{"key":"2025120902301476000_2025.12.04.692467v1.10","doi-asserted-by":"publisher","DOI":"10.1038\/nature13489"},{"key":"2025120902301476000_2025.12.04.692467v1.11","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1800438"},{"key":"2025120902301476000_2025.12.04.692467v1.12","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2010.01.040"},{"key":"2025120902301476000_2025.12.04.692467v1.13","doi-asserted-by":"publisher","DOI":"10.1038\/s41577-023-00849-x"},{"key":"2025120902301476000_2025.12.04.692467v1.14","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.248344"},{"key":"2025120902301476000_2025.12.04.692467v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2022.10.002"},{"key":"2025120902301476000_2025.12.04.692467v1.16","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0012320"},{"key":"2025120902301476000_2025.12.04.692467v1.17","doi-asserted-by":"publisher","DOI":"10.1002\/eji.201141548"},{"key":"2025120902301476000_2025.12.04.692467v1.18","doi-asserted-by":"publisher","DOI":"10.1111\/j.1462-5822.2011.01625.x"},{"key":"2025120902301476000_2025.12.04.692467v1.19","doi-asserted-by":"publisher","DOI":"10.1111\/j.1462-5822.2010.01450.x"},{"key":"2025120902301476000_2025.12.04.692467v1.20","doi-asserted-by":"crossref","unstructured":"Szczerba M , Ganesh A , Gil-Marqu\u00e9s ML , Briken V , Goldberg MB . 2025. 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