A Bacterial Effector Reveals the V-ATPase-ATG16L1 Axis that Initiates Xenophagy.

Antibacterial autophagy (xenophagy) is an important host defense, but how it is initiated is unclear. Here, we performed a bacterial transposon screen and identified a T3SS effector SopF that potently blocked Salmonella autophagy. SopF was a general xenophagy inhibitor without affecting canonical autophagy. S. Typhimurium ΔsopF resembled S. flexneri ΔvirAΔicsB with the majority of intracellular bacteria targeted by autophagy, permitting a CRISPR screen that identified host V-ATPase as an essential factor. Upon bacteria-caused vacuolar damage, the V-ATPase recruited ATG16L1 onto bacteria-containing vacuole, which was blocked by SopF. Mammalian ATG16L1 bears a WD40 domain required for interacting with the V-ATPase. Inhibiting autophagy by SopF promoted S. Typhimurium proliferation in vivo. SopF targeted Gln124 of ATP6V0C in the V-ATPase for ADP-ribosylation. Mutation of Gln124 also blocked xenophagy, but not canonical autophagy. Thus, the discovery of SopF reveals the V-ATPase-ATG16L1 axis that critically mediates autophagic recognition of intracellular pathogen.

Legionella effector LnaB is a phosphoryl-AMPylase that impairs phosphosignalling

AMPylation is a post-translational modification in which AMP is added to the amino acid side chains of proteins1,2. Here we show that, with ATP as the ligand and actin as the host activator, the effector protein LnaB of Legionella pneumophila exhibits AMPylase activity towards the phosphoryl group of phosphoribose on PRR42-Ub that is generated by the SidE family of effectors, and deubiquitinases DupA and DupB in an E1- and E2-independent ubiquitination process3-7. The product of LnaB is further hydrolysed by an ADP-ribosylhydrolase, MavL, to Ub, thereby preventing the accumulation of PRR42-Ub and ADPRR42-Ub and protecting canonical ubiquitination in host cells. LnaB represents a large family of AMPylases that adopt a common structural fold, distinct from those of the previously known AMPylases, and LnaB homologues are found in more than 20 species of bacterial pathogens. Moreover, LnaB also exhibits robust phosphoryl AMPylase activity towards phosphorylated residues and produces unique ADPylation modifications in proteins. During infection, LnaB AMPylates the conserved phosphorylated tyrosine residues in the activation loop of the Src family of kinases8,9, which dampens downstream phosphorylation signalling in the host. Structural studies reveal the actin-dependent activation and catalytic mechanisms of the LnaB family of AMPylases. This study identifies, to our knowledge, an unprecedented molecular regulation mechanism in bacterial pathogenesis and protein phosphorylation.

Threonine ADP-Ribosylation of Ubiquitin by a Bacterial Effector Family Blocks Host Ubiquitination.

Ubiquitination is essential for numerous eukaryotic cellular processes. Here, we show that the type III effector CteC from Chromobacterium violaceum functions as an adenosine diphosphate (ADP)-ribosyltransferase that specifically modifies ubiquitin via threonine ADP-ribosylation on residue T66. The covalent modification prevents the transfer of ubiquitin from ubiquitin-activating enzyme E1 to ubiquitin-conjugating enzyme E2, which inhibits subsequent ubiquitin activation by E2 and E3 enzymes in the ubiquitination cascade and leads to the shutdown of polyubiquitin synthesis in host cells. This unique modification also causes dysfunction of polyubiquitin chains in cells, thereby blocking host ubiquitin signaling. The disruption of host ubiquitination by CteC plays a crucial role in C. violaceum colonization in mice during infection. CteC represents a family of effector proteins in pathogens of hosts from different kingdoms. All the members of this family specifically ADP-ribosylate ubiquitin. The action of CteC reveals a new mechanism for interfering with host ubiquitination by pathogens.

A Type I-F Anti-CRISPR Protein Inhibits the CRISPR-Cas Surveillance Complex by ADP-Ribosylation.

CRISPR-Cas systems are bacterial anti-viral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here, we report a novel mechanism by which AcrIF11 inhibits the type I-F CRISPR system. Our structural and biochemical studies demonstrate that AcrIF11 functions as a novel mono-ADP-ribosyltransferase (mART) to modify N250 of the Cas8f subunit, a residue required for recognition of the protospacer-adjacent motif, within the crRNA-guided surveillance (Csy) complex from Pseudomonas aeruginosa. The AcrIF11-mediated ADP-ribosylation of the Csy complex results in complete loss of its double-stranded DNA (dsDNA) binding activity. Biochemical studies show that AcrIF11 requires, besides Cas8f, the Cas7.6f subunit for binding to and modifying the Csy complex. Our study not only reveals an unprecedented mechanism of type I CRISPR-Cas inhibition and the evolutionary arms race between phages and bacteria but also suggests an approach for designing highly potent regulatory tools in the future applications of type I CRISPR-Cas systems.

Shigella evades pyroptosis by arginine ADP-riboxanation of caspase-11.

Mouse caspase-11 and human caspase-4 and caspase-5 recognize cytosolic lipopolysaccharide (LPS) to induce pyroptosis by cleaving the pore-forming protein GSDMD1-5. This non-canonical inflammasome defends against Gram-negative bacteria6,7. Shigella flexneri, which causes bacillary dysentery, lives freely within the host cytosol where these caspases reside. However, the role of caspase-11-mediated pyroptosis in S. flexneri infection is unknown. Here we show that caspase-11 did not protect mice from S. flexneri infection, in contrast to infection with another cytosolic bacterium, Burkholderia thailandensis8. S. flexneri evaded pyroptosis mediated by caspase-11 or caspase 4 (hereafter referred to as caspase-11/4) using a type III secretion system (T3SS) effector, OspC3. OspC3, but not its paralogues OspC1 and 2, covalently modified caspase-11/4; although it used the NAD+ donor, this modification was not ADP-ribosylation. Biochemical dissections uncovered an ADP-riboxanation modification on Arg314 and Arg310 in caspase-4 and caspase-11, respectively. The enzymatic activity was shared by OspC1 and 2, whose ankyrin-repeat domains, unlike that of OspC3, could not recognize caspase-11/4. ADP-riboxanation of the arginine blocked autoprocessing of caspase-4/11 as well as their recognition and cleavage of GSDMD. ADP-riboxanation of caspase-11 paralysed pyroptosis-mediated defence in Shigella-infected mice and mutation of ospC3 stimulated caspase-11- and GSDMD-dependent anti-Shigella humoral immunity, generating a vaccine-like protective effect. Our study establishes ADP-riboxanation of arginine as a bacterial virulence mechanism that prevents LPS-induced pyroptosis.

Acetylation of PHF5A Modulates Stress Responses and Colorectal Carcinogenesis through Alternative Splicing-Mediated Upregulation of KDM3A.

Alternative pre-mRNA-splicing-induced post-transcriptional gene expression regulation is one of the pathways for tumors maintaining proliferation rates accompanying the malignant phenotype under stress. Here, we uncover a list of hyperacetylated proteins in the context of acutely reduced Acetyl-CoA levels under nutrient starvation. PHF5A, a component of U2 snRNPs, can be acetylated at lysine 29 in response to multiple cellular stresses, which is dependent on p300. PHF5A acetylation strengthens the interaction among U2 snRNPs and affects global pre-mRNA splicing pattern and extensive gene expression. PHF5A hyperacetylation-induced alternative splicing stabilizes KDM3A mRNA and promotes its protein expression. Pathologically, PHF5A K29 hyperacetylation and KDM3A upregulation axis are correlated with poor prognosis of colon cancer. Our findings uncover a mechanism of an anti-stress pathway through which acetylation on PHF5A promotes the cancer cells' capacity for stress resistance and consequently contributes to colon carcinogenesis.

Molecular basis of threonine ADP-ribosylation of ubiquitin by bacterial ARTs.

Ubiquitination plays essential roles in eukaryotic cellular processes. The effector protein CteC from Chromobacterium violaceum blocks host ubiquitination by mono-ADP-ribosylation of ubiquitin (Ub) at residue T66. However, the structural basis for this modification is unknown. Here we report three crystal structures of CteC in complexes with Ub, NAD+ or ADP-ribosylated Ub, which represent different catalytic states of CteC in the modification. CteC adopts a special 'D-E' catalytic motif for catalysis and binds NAD+ in a half-ligand binding mode. The specific recognition of Ub by CteC is determined by a relatively separate Ub-targeting domain and a long loop L6, not the classic ADP-ribosylating turn-turn loop. Structural analyses with biochemical results reveal that CteC represents a large family of poly (ADP-ribose) polymerase (PARP)-like ADP-ribosyltransferases, which harbors chimeric features from the R-S-E and H-Y-E classes of ADP-ribosyltransferases. The family of CteC-like ADP-ribosyltransferases has a common 'D-E' catalytic consensus and exists extensively in bacteria and eukaryotic microorganisms.

Dps-dependent in vivo mutation enhances long-term host adaptation in Vibrio cholerae.

As one of the most successful pathogenic organisms, Vibrio cholerae (V. cholerae) has evolved sophisticated regulatory mechanisms to overcome host stress. During long-term colonization by V. cholerae in adult mice, many spontaneous nonmotile mutants (approximately 10% at the fifth day post-infection) were identified. These mutations occurred primarily in conserved regions of the flagellar regulator genes flrA, flrC, and rpoN, as shown by Sanger and next-generation sequencing, and significantly increased fitness during colonization in adult mice. Intriguingly, instead of key genes in DNA repair systems (mutS, nfo, xthA, uvrA) or ROS and RNS scavenging systems (katG, prxA, hmpA), which were generally thought to be associated with bacterial mutagenesis, we found that deletion of the cyclin gene dps significantly increased the mutation rate (up to 53% at the fifth day post-infection) in V. cholerae. We further determined that the dpsD65A and dpsF46E point mutants showed a similar mutagenesis profile as the Δdps mutant during long-term colonization in mice, which strongly indicated that the antioxidative function of Dps directly contributes to the development of V. cholerae nonmotile mutants. Methionine metabolism pathway may be one of the mechanism for ΔflrA, ΔflrC and ΔrpoN mutant increased colonization in adult mice. Our results revealed a new phenotype in which increased fitness of V. cholerae in the host gut via spontaneous production nonmotile mutants regulated by cyclin Dps, which may represent a novel adaptation strategy for directed evolution of pathogens in the host.

Legionella effectors SidC/SdcA ubiquitinate multiple small GTPases and SNARE proteins to promote phagosomal maturation.

Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.

The Salmonella Typhimurium Effector SpvB Subverts Host Membrane Trafficking by Targeting Clathrin and AP-1.

Salmonella enterica, the etiological agent of gastrointestinal and systemic diseases, translocates a plethora of virulence factors through its type III secretion systems to host cells during infection. Among them, SpvB has been reported to harbor an ADP-ribosyltransferase domain in its C terminus, which destabilizes host cytoskeleton by modifying actin. However, whether this effector targets other host factors as well as the function of its N terminus still remains to be determined. Here, we found that SpvB targets clathrin and its adaptor AP-1 (adaptor protein 1) via interactions with its N-terminal domain. Notably, our data suggest that SpvB-clathrin/AP-1 associations disrupt clathrin-mediated endocytosis and protein secretion pathway as well. In addition, knocking down of AP-1 promotes Salmonella intracellular survival and proliferation in host cells.

S-Nitrosylation of the histone deacetylase HDA19 stimulates its activity to enhance plant stress tolerance in Arabidopsis.

Arabidopsis histone deacetylase HDA19 is required for gene expression programs of a large spectrum of plant developmental and stress-responsive pathways. How this enzyme senses cellular environment to control its activity remains unclear. In this work, we show that HDA19 is post-translationally modified by S-nitrosylation at 4 Cysteine (Cys) residues. HDA19 S-nitrosylation depends on the cellular nitric oxide level, which is enhanced under oxidative stress. We find that HDA19 is required for cellular redox homeostasis and plant tolerance to oxidative stress, which in turn stimulates its nuclear enrichment, S-nitrosylation and epigenetic functions including binding to genomic targets, histone deacetylation and gene repression. The Cys137 of the protein is involved in basal and stress-induced S-nitrosylation, and is required for HDA19 functions in developmental, stress-responsive and epigenetic controls. Together, these results indicate that S-nitrosylation regulates HDA19 activity and is a mechanism of redox-sensing for chromatin regulation of plant tolerance to stress.

The ever-increasing necessity of mass spectrometry in dissecting protein post-translational modifications catalyzed by bacterial effectors.

Protein post-translational modifications (PTMs), such as ADP-ribosylation and phosphorylation, regulate multiple fundamental biological processes in cells. During bacterial infection, effector proteins are delivered into host cells through dedicated bacterial secretion systems and can modulate important cellular pathways by covalently modifying their host targets. These strategies enable intruding bacteria to subvert various host processes, thereby promoting their own survival and proliferation. Despite rapid expansion of our understanding of effector-mediated PTMs in host cells, analytical measurements of these molecular events still pose significant challenges in the study of host-pathogen interactions. Nevertheless, with major technical breakthroughs in the last two decades, mass spectrometry (MS) has evolved to be a valuable tool for detecting protein PTMs and mapping modification sites. Additionally, large-scale PTM profiling, facilitated by different enrichment strategies prior to MS analysis, allows high-throughput screening of host enzymatic substrates of bacterial effectors. In this review, we summarize the advances in the studies of two representative PTMs (i.e., ADP-ribosylation and phosphorylation) catalyzed by bacterial effectors during infection. Importantly, we will discuss the ever-increasing role of MS in understanding these molecular events and how the latest MS-based tools can aid in future studies of this booming area of pathogenic bacteria-host interactions.

Glycemic Index, Glycemic Load, and Cardiovascular Disease and Mortality.

BACKGROUND: Most data regarding the association between the glycemic index and cardiovascular disease come from high-income Western populations, with little information from non-Western countries with low or middle incomes. To fill this gap, data are needed from a large, geographically diverse population. METHODS: This analysis includes 137,851 participants between the ages of 35 and 70 years living on five continents, with a median follow-up of 9.5 years. We used country-specific food-frequency questionnaires to determine dietary intake and estimated the glycemic index and glycemic load on the basis of the consumption of seven categories of carbohydrate foods. We calculated hazard ratios using multivariable Cox frailty models. The primary outcome was a composite of a major cardiovascular event (cardiovascular death, nonfatal myocardial infarction, stroke, and heart failure) or death from any cause. RESULTS: In the study population, 8780 deaths and 8252 major cardiovascular events occurred during the follow-up period. After performing extensive adjustments comparing the lowest and highest glycemic-index quintiles, we found that a diet with a high glycemic index was associated with an increased risk of a major cardiovascular event or death, both among participants with preexisting cardiovascular disease (hazard ratio, 1.51; 95% confidence interval [CI], 1.25 to 1.82) and among those without such disease (hazard ratio, 1.21; 95% CI, 1.11 to 1.34). Among the components of the primary outcome, a high glycemic index was also associated with an increased risk of death from cardiovascular causes. The results with respect to glycemic load were similar to the findings regarding the glycemic index among the participants with cardiovascular disease at baseline, but the association was not significant among those without preexisting cardiovascular disease. CONCLUSIONS: In this study, a diet with a high glycemic index was associated with an increased risk of cardiovascular disease and death. (Funded by the Population Health Research Institute and others.).

Genome-wide identification of alcohol dehydrogenase (ADH) gene family in oilseed rape (Brassica napus L.) and BnADH36 functional verification under salt stress.

BACKGROUND: Alcohol dehydrogenase (ADH) is an enzyme that binds to zinc, facilitating the interconversion of ethanol and acetaldehyde or other corresponding alcohols/aldehydes in the pathway of ethanol fermentation. It plays a pivotal role in responding to environmental stress. However, the response of the ADH family to abiotic stress remains unknown in rapeseed. RESULT: In this study, we conducted a comprehensive genome-wide investigation of the ADH family in rapeseed, encompassing analysis of their gene structure, replication patterns, conserved motifs, cis-acting elements, and response to stress. A total of 47 ADH genes were identified within the rapeseed genome. Through phylogenetic analysis, BnADHs were classified into four distinct clades (I, II, IV, V). Prediction of protein domains revealed that all BnADH members possessed a GroES-like (ADH_N) domain and a zinc-bound (ADH_zinc_N) domain. Analysis of promoter sequences demonstrated that BnADHs contained numerous cis-acting elements associated with hormone and stress responses, indicating their widespread involvement in various biological regulatory processes. Expression profiling under different concentrations of salt stress treatments (0%, 0.4%, 0.8%, 1.0% NaCl) further highlighted the significant role played by the BnADH family in abiotic stress response mechanisms. Overexpression of BnADH36 in rapeseed significantly improved the salt tolerance of rapeseed. CONCLUSION: The features of the BnADH family in rapeseed was comprehensively characterized in this study, which could provide reference to the research of BnADHs in abiotic stress response.

Exercise Prescription in Individuals with Prehypertension and Hypertension: Systematic Review and Meta-Analysis.

Background: The prevalence of prehypertension and hypertension has been increasing over the years, and is closely related to cardiovascular and cerebrovascular diseases. Exercise is an effective method of lifestyle intervention, and it aims to lower blood pressure and control other risks. Studies have shown that different modes of exercise have varying effects on blood pressure, and individuals with prehypertension or hypertension need to carry out this intervention by using personalized modes of exercise. Methods: We conducted a systematic review and meta-analysis to evaluate the effects of different modes of exercise regimens on systolic blood pressure, diastolic blood pressure and heart rate in individuals with high-normal blood pressure and hypertension. We included 27 trials, and 2731 individuals were under 8 exercise regimens. Stata12.0 statistical software was used for statistical analysis. Results: Heat pools significantly reduced systolic blood pressure (SBP) by 15.62 mmHg (95% confidence interval [CI]: -23.83, -7.41), and cycling reduced SBP by 14.76 mmHg (-17.04, -12.48). Two to three types of aerobic exercise performed at the same time also significantly reduced diastolic blood pressure (DBP) by 5.61 mmHg (-7.71, -3.52), and isometric handgrip training exercise reduced DBP by 5.57 mmHg (-7.48, -3.66). Cycling also significantly reduced heart rate (HR) by 9.57 beats/minute (-11.25, -7.90). Conclusions: The existing literature suggests that different types of exercise can effectively reduce the levels of SBP, DBP and HR in individuals with prehypertension or hypertension.

Brain frailty associated with stroke events in anterior circulation large artery occlusion.

OBJECTIVE: To investigate the factors associated with brain frailty and the effect of brain frailty in patients with anterior circulation large artery occlusion (AC-LAO). METHODS: 1100 patients with AC-LVO consecutively admitted to the Second Hospital of Hebei Medical University, North China between June 2016 and April 2018 were retrospectively analyzed. The variables associated with brain frailty and stroke outcome were analyzed by ANOVA analysis, the Mann-Whitney U test and multiple linear regression. Based on previous research. Brain frailty score comprises 1 point each for white matter hyperintensity (WMH), old infarction lesions, and cerebral atrophy among 983 participants with baseline brain magnetic resonance imaging or computed tomography. RESULTS: Among AC-LAO participants, baseline brain frailty score ≥ 1 was common (750/983, 76.3%). Duration of hypertension > 5 years (mean difference [MD] 0.236, 95% CI 0.077, 0.395, p = 0.004), multiple vessel occlusion (MD 0.339, 95% CI 0.068, 0.611, p = 0.014) and basal ganglia infarction (MD -0.308, 95% CI -0.456, -0.160, p < 0.001) were independently associated with brain frailty score. Brain frailty score was independently associated with stroke events, and higher brain frailty scores were associated with higher rates of stroke events (p < 0.001). However, brain frailty has no independent effect on short-term outcome of ACI in AC-LAO patients. CONCLUSIONS: In AC-LAO patients, older age, duration of hypertension > 5 years, and multiple vessel occlusion influenced the brain frailty score. Brain frailty score was independently associated with the occurrence of stroke events in AC-LAO patients.

Structural basis of ubiquitin modification by the Legionella effector SdeA.

Protein ubiquitination is a multifaceted post-translational modification that controls almost every process in eukaryotic cells. Recently, the Legionella effector SdeA was reported to mediate a unique phosphoribosyl-linked ubiquitination through successive modifications of the Arg42 of ubiquitin (Ub) by its mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains. However, the mechanisms of SdeA-mediated Ub modification and phosphoribosyl-linked ubiquitination remain unknown. Here we report the structures of SdeA in its ligand-free, Ub-bound and Ub-NADH-bound states. The structures reveal that the mART and PDE domains of SdeA form a catalytic domain over its C-terminal region. Upon Ub binding, the canonical ADP-ribosyltransferase toxin turn-turn (ARTT) and phosphate-nicotinamide (PN) loops in the mART domain of SdeA undergo marked conformational changes. The Ub Arg72 might act as a 'probe' that interacts with the mART domain first, and then movements may occur in the side chains of Arg72 and Arg42 during the ADP-ribosylation of Ub. Our study reveals the mechanism of SdeA-mediated Ub modification and provides a framework for further investigations into the phosphoribosyl-linked ubiquitination process.

Feminization of the health workforce in China: exploring gendered composition from 2002 to 2020.

BACKGROUND: Feminization of health workforce has been globally documented, but it has not been investigated in China. This study aims to analyze changes in the gendered composition of health workforce and explore the trend in different types of health workforce, health organizations and majors within China's health system. METHODS: The data were collected from China Health Statistical Yearbook from 2002 to 2020. We focused on health professionals including doctors, nurses, and pharmacists in health organizations. Trend analysis was employed to examine the change in the ratio of female health workforce over 18 years. The estimated average annual percent change (AAPC) was estimated, and the reciprocals of variances for the female ratios were used as weights. RESULTS: In China, health professionals increased from 4.7 million in 2002 to 10.68 million in 2020. Health professionals per 1000 population increased from 3.41 in 2002 to 7.57 in 2020. The ratio of female health professionals significantly increased from 63.85% in 2002 to 72.4% in 2020 (AAPC = 1.04%, 95% CI 0.96-1.11%, P < 0.001). Female doctors and pharmacists increased 4.7 and 7.9 percentage points from 2002 to 2020. Female health workers at township health centers, village clinics, centers for disease control and prevention had higher annual increase rate (AAPC = 1.67%, 2.25% and 1.33%, respectively) than those at hospital (0.70%) and community health center (0.5%). Female doctors in traditional Chinese medicine, dentistry and public health had higher annual increase rate (AAPC = 1.82%, 1.53% and 1.91%, respectively) than female clinical doctor (0.64%). CONCLUSIONS: More women are participating in the healthcare sector in China. However, socially lower-ranked positions have been feminizing faster, which could be due to the inherent and structural gender norms restricting women's career. More collective and comprehensive system-level actions will be needed to foster a gender-equitable environment for health workforce at all levels.

Gender income differences among general practitioners with compulsory services in early career stage in underdeveloped areas: evidence from a prospective cohort study in China.

BACKGROUND: Gender equality and the gender income gap in medicine are long-standing global problems. Although gender-related differences have been widely studied in developed countries, they remain unclear in underdeveloped regions. In 2010, China initiated a national compulsory service program (CSP) to train qualified general practitioners in rural and remote areas. This study aimed to evaluate gender income differences for early career CSP and non-CSP (NCSP) graduates in underdeveloped areas. METHODS: A cohort study was conducted with 3620 CSP and NCSP graduates from four medical universities in Central and Western China. Baseline surveys and six follow-up surveys were conducted between 2015 and 2022. Incomes, including monthly mean income and proportion of performance-based income, were measured as the key outcome variables. Multivariate linear regression models were used to identify the gender income gap. RESULTS: NCSP graduates had higher average monthly incomes than CSP graduates. In the seventh year after graduation, the average monthly income for NCSP graduates was 7859 CNY while was 5379 CNY for CSP graduates. After controlling for demographic characteristics, the gender monthly income gap for CSP graduates was expanded from the fourth year (3.0%) to the sixth year (5.9%) after graduation, and that for NCSP graduates was expanded from the fifth year (11.9%) to the seventh year (16.3%) after graduation. Regarding performance-based income, it was 58.9% for NCSP graduates and 45.8% for CSP graduates in the seventh year after graduation. After controlling for performance-based income proportion, the gender income gap was reduced from 5.9 to 4.0% in the sixth year after graduation for CSP graduates, and from 16.3 to 14.4% for NCSP graduates in the seventh year after graduation. CONCLUSION: An extensive and ever-increasing gender income gap exists among young doctors in the early stages of their careers in underdeveloped areas of China. The high proportion of performance-based income among men is one of the main explanations for the observed difference. A more explicit compensation system must be established to enhance support for female health workers.