Sabatino, B. | Search bioRxiv

bioRxiv2023

Dissecting components of the Campylobacter jejuni fetMP-fetABCDEF gene cluster in iron scavenging

Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Acute infection can be antecedent to highly debilitating long-term sequelae. Expression of iron acquisition systems is vital for C. jejuni to survive the low iron availability within the human gut. The C. jejuni fetMP-fetABCDEF gene cluster is known to be upregulated during human infection and under iron limitation. While FetM and FetP have been functionally linked to iron transport in prior work, here we assess the contribution by each of the downstream genes (fetABCDEF) to C. jejuni growth during both iron-depleted and iron-replete conditions. Significant growth impairment was observed upon disruption of fetA, fetB, fetC, and fetD, suggesting a role in iron acquisition for each encoded protein. FetA expression was modulated by iron-availability but not dependent on the presence of FetB, FetC, FetD, FetE or FetF. Functions of the putative thioredoxins FetE and FetF were redundant in iron scavenging, requiring a double deletion ({Delta}fetEF) to exhibit a growth defect. C. jejuni FetE was expressed and the structure solved to 1.50 [A], revealing structural similarity to thiol-disulfide oxidases. Functional characterization in biochemical assays showed that FetE reduced insulin at a slower rate than E. coli Trx and that together, FetEF promoted substrate oxidation in cell extracts, suggesting that FetE (and presumably FetF) are oxidoreductases that can mediate oxidation in vivo. This study advances our understanding of the contributions by the fetMP-fetABCDEF gene cluster to virulence at a genetic and functional level, providing foundational knowledge towards mitigating C. jejuni-related morbidity and mortality.

microbiology↗

bioRxiv2023

The unfolded protein response of the endoplasmic reticulum protects Caenorhabditis elegans against DNA damage caused by stalled replication forks

All animals must maintain genome and proteome integrity, especially when experiencing endogenous or exogenous stress. To cope, organisms have evolved sophisticated and conserved response systems: unfolded protein responses (UPRs) ensure proteostasis while DNA damage responses (DDRs) maintains genome integrity. Emerging evidence suggests that UPRs and DDRs crosstalk, but this remains poorly understood. Here, we demonstrate that depletion of the DNA primases pri-1 or pri-2, which synthesize RNA primers at replication forks and whose inactivation causes DNA damage, activates the UPR of the endoplasmic reticulum (UPR-ER) in Caenorhabditis elegans, with especially strong activation in the germline. We observed activation of both the inositol-requiring-enzyme 1 (ire-1) and the protein kinase RNA-like ER kinase (pek-1) branches of the UPR-ER. Interestingly, activation of the UPR-ER output gene heat shock protein 4 (hsp-4) was partially independent of its canonical activators, ire-1 and X-box binding protein (xbp-1), and instead required the third branch of the UPR-ER, activating transcription factor 6 (atf-6), suggesting functional redundancy. We further found that primase depletion specifically induces the UPR-ER, but not the distinct cytosolic or mitochondrial UPRs, suggesting that primase inactivation causes compartment-specific rather than global stress. Functionally, loss of ire-1 or pek-1 sensitized animals to replication stress caused by hydroxyurea. Finally, transcriptome analysis of pri-1 embryos revealed several deregulated processes that could cause UPR-ER activation, including protein glycosylation, calcium signaling, and fatty acid desaturation. Together, our data show that the UPR-ER, but not other UPRs, responds to replication fork stress and that the UPR-ER is required to alleviate this stress.

genetics↗