Huizhou Fan

Bacterial survival under environmental stress requires precise regulation of protein homeostasis, yet how this is achieved in organisms with streamlined genomes remains incompletely understood. Here, we identify HagF as a previously unrecognized protein that antagonizes the conserved heat-inducible transcriptional repressor HrcA in Chlamydia . Using genetic and biochemical approaches, including overexpression and CRISPRi-mediated knockdown, we show that under non-stressed conditions, HrcA represses hagF along with its five known targets, including hrcA and four chaperone genes. Upon heat stress, HagF is induced and binds HrcA, attenuating its repressor activity. This negative feedback loop enhances chaperone gene induction, particularly from the hrcA-grpE-dnaK operon, and promotes infectious elementary body yield. Notably, HagF is conserved across members of the order Chlamydiales , highlighting a lineage-specific adaptation. Together, these findings define a regulatory circuit in which a specialized antagonist modulates a widely conserved repressor, enabling Chlamydia to couple heat-responsive gene expression with developmental progression and enhance fitness under restrictive conditions.

We describe an oligonucleotide RNA-guided, DNase-dead CRISPR-associated protein 12 (dCas12)-mediated transcriptional interference (CRISPRi) system optimized for gene knockdown in Chlamydia trachomatis. In this system, while guide RNAs are expressed constitutively, dCas12 expression is conditional and depends on the presence of the small molecule anhydrotetracycline (ATC). This design enables temporal control of gene repression, allowing the functional analysis of a wide range of genes, including essential genes, without permanent genetic disruption. Due to its T-rich protospacer adjacent motif (PAM) requirement, dCas12 is particularly well-suited for targeting genes in the highly A/T-rich genomes of pathogenic chlamydiae. We further detail strategies for genetic complementation through co-expression of dCas12-resistant alleles, thereby permitting validation of knockdown phenotypes.

We describe in detail an experimental strategy, DOPE (Dependence On Plasmid-mediated Expression), to enable disruption of an essential chlamydial gene. A wild-type Chlamydia trachomatis strain is first transformed with a β-lactamase-selectable plasmid carrying a copy of the gene under the inducible control of an anhydrotetracycline (ATC)-responsive promoter. Transformants are selected under permissive conditions in the absence of ATC to establish stable plasmid maintenance. The resulting chlamydiae are subsequently transformed with a suicide plasmid encoding a group II intron engineered to target the chromosomal copy of the gene. Secondary transformants with the intron inserted into the chromosomal locus are then selected with spectinomycin in the presence of ATC to induce expression of the plasmid-encoded allele. This protocol thus produces a conditional knockout strain in which expression of the essential gene is inducibly controlled by ATC so that its function in chlamydial growth and developmental progression can be delineated.

The developmental cycle of Chlamydia trachomatis requires coordinated transitions between infectious elementary bodies (EBs) and replicative reticulate bodies (RBs), yet the temporal organization of the underlying transcriptional programs remains incompletely resolved. We performed high-coverage RNA sequencing from 0 to 32 h post-infection (hpi), with closely spaced early sampling, and used unsupervised clustering of temporal expression profiles to identify four coordinated programs: immediate-early host adaptation and developmental program, RB formation and maintenance, RB proliferation and secondary differentiation, and EB formation and invasion. The immediate-early program peaked at 1 hpi and contained a strong representation of inclusion-membrane and intracellular-trafficking genes, together with the developmental regulator gene euo. The RB formation and maintenance program peaked at approximately 3 hpi and sustained expression throughout much of the cycle, encompassing genes that support transcription, translation, and core metabolism. The RB proliferation and secondary differentiation program combined DNA replication and cell-division genes with alternative sigma factors, partner-switching components, and type III secretion apparatus genes, consistent with developmental preparation during proliferation. The EB formation and invasion program contained genes associated with structural maturation, host-cell interaction, and infectious competence. These findings distinguish transient host-adaptation expression from sustained early biosynthetic expression, separate RB establishment from population expansion, and differentiate regulatory preparation for the RB-to-EB transition from later EB maturation. Together, the four programs define partially overlapping transcriptional priorities that provide a framework for investigating how gene expression coordinates chlamydial growth, differentiation, and preparation for the next infection cycle.

Following an intravaginal inoculation with Chlamydia trachomatis , mice deficient in type I interferon receptor IFNαR1 (IFNαR1 ^-/- ) significantly increased the yield of live chlamydiae on days 3 & 5 but reduced it to the level of wild-type mice by day 7, while mice deficient in type II interferon receptor IFNγR1 (IFNγR1 ^-/- ) significantly increased the chlamydial yield by day 5 and the increase persisted throughout the remainder of the infection course. These observations reveal a temporal division of labor between type I & II interferons in regulating C. trachomatis infection in the female genital tract. Interestingly, mice deficient in both IFNαR1 & IFNγR1 exhibited higher mortality and shed more chlamydial organisms than IFNγR1 ^-/- mice by week 6, suggesting that IFNαR1 remains critical for inhibiting C. trachomatis at late stages. An anti-IFNαR1 antibody blockade significantly increased chlamydial yields in IFNγR1 ^-/- mice, suggesting that the anti-chlamydial activities of type I & II interferon systems are both distinct and overlapping throughout the infection course. Furthermore, the anti-chlamydial activity of type I interferon signaling is localized to the lower vagina, while that of type II interferon signaling is localized to the upper vagina. Thus, we have demonstrated that type I & II interferons function complementarily and synergistically in time and space to control C. trachomatis infection, laying the foundation for further elucidating the mechanisms of IFN regulation of chlamydial infection and for developing interventional and preventive strategies against C. trachomatis in the female genital tract. Importance: Lack of information on the precise roles of type I and type II interferons during chlamydial infection has hindered the development of interferon-based strategies to prevent chlamydial infection and pathogenicity. The current study has revealed a temporal division of labor between type I & type II interferons in regulating chlamydial infection in the female genital tract, with type I acting earlier than type II during the innate phase. Nevertheless, type I remains critical for cooperating with type II to suppress chlamydia 6 weeks after infection. Finally, type I interferons seem to mainly target chlamydial infection in the lower vagina, while type II interferons target the infection in the upper vagina. These new findings on the distinct and overlapping roles of type I and type II interferons in regulating chlamydial infection may guide the development of interferon-based precision strategies to reduce chlamydial infection and pathogenicity in the female genital tract.

The unique chlamydial developmental cycle comprises three stages: primary differentiation of infectious elementary bodies (EBs) into reticulate bodies (RBs), RB replication, and secondary differentiation into progeny EBs. Extensive chromosome remodeling during RB-to-EB differentiation is thought to be mediated by the histone homologs HctA and HctB. Here, we used an inducible CRISPR interference system to knock down hctA , hctB , or both genes in Chlamydia trachomatis . Surprisingly, knockdown of either histone gene alone or in combination caused only modest reductions in EB yield and did not prevent nucleoid condensation during the parental developmental cycle. In contrast, progeny EBs generated under histone knockdown conditions were markedly impaired in their ability to initiate subsequent infections. Secondary cultures initiated with HctA-deficient EBs exhibited a delay in primary EB-to-RB differentiation, whereas combined repression of hctA and hctB caused both delayed differentiation and persistently reduced genome accumulation, indicative of impaired RB formation and subsequent growth. Repression of hctB alone did not measurably affect genome replication in secondary cultures. Together, these findings reveal that chromosome organization established during EB maturation governs developmental fitness in the next infection cycle, uncovering a transgenerational role for chlamydial histone homologs. IMPORTANCE: Chlamydial histone homologs HctA and HctB are unusual among bacterial DNA-binding proteins in that they share sequence homology with mammalian histones and are developmentally regulated during the formation of infectious particles. Here, we show that reduced expression of HctA and HctB has only modest effects on visible nucleoid condensation and EB production, consistent with partial functional redundancy between the two histones and suggesting that additional nucleoid factors contribute to EB chromosome compaction. In contrast, deficiency of HctA and HctB during EB maturation has profound consequences in the next infection cycle, impairing completion of primary EB-to-RB differentiation and subsequent RB growth. These findings reveal a previously unrecognized transgenerational role for chlamydial histone homologs and establish chromosome organization during EB maturation as a key determinant of developmental fitness across infection cycles.

Chlamydia trachomatis is a leading cause of urogenital infections that can result in serious long-term complications. This obligate intracellular bacterium undergoes a biphasic developmental cycle alternating between the infectious elementary body and the replicative reticulate body and can enter a persistent state in response to adverse environmental conditions. Although transcriptomic reprogramming is central to chlamydial stress adaptation and persistence, how responses differ across biologically distinct stressors remains incompletely defined. Here, we performed a comparative reanalysis of five published, high-quality C. trachomatis RNA-Seq data sets generated under prolonged interferon-γ treatment, tryptophan starvation, iron starvation, penicillin exposure, or acute heat shock. Global transcriptomic analyses reveal stress-specific reprogramming and a clear separation between the transcriptome induced by heat shock and those induced by chronic stresses. Transcriptomic overlap observed among chronic stress conditions is substantially reduced when the heat shock transcriptome is included, indicating that shared transcriptional features are stressor-dependent. Consistent with prior findings, tryptophan starvation and iron starvation exhibit particularly close transcriptomic similarity, likely reflecting regulatory cross-talk mediated by the iron-dependent transcriptional repressor YtgR. Notably, this similarity exceeds that observed between tryptophan starvation and interferon-γ treatment, despite the well-established role of interferon-γ in inducing host-mediated tryptophan depletion. In contrast, interferon-γ induces a distinct but partially overlapping transcriptome, likely reflecting activation of additional host-mediated antimicrobial mechanisms beyond tryptophan deprivation. Together, these findings demonstrate that adaptation to different biological stressors in C. trachomatis is driven by distinct transcriptomic reprogramming, while consistently involving a subset of functions that may represent points of vulnerability for disrupting chlamydial persistence.

Sexually transmitted infection with Chlamydia trachomatis can cause pathology, such as hydrosalpinx, in the upper genital tract, leading to infertility. To investigate how genetic variation affects chlamydial pathogenicity, we screened five strains of Collaborative Cross (CC) mice for susceptibility to intravaginal infection and hydrosalpinx induction by C. muridarum , a mouse-adapted chlamydial species used extensively to reveal chlamydial pathogenic mechanisms. In terms of susceptibility to genital chlamydial infection, the five CC strains fell into two categories: CC011 and CC012 were resistant, while CC037, CC042, and CC080 were susceptible. The resistant strains shed significant levels of live organisms from the genital tract for only ~2 weeks, whereas the susceptible strains shed for ~4 weeks. However, the resistant CC012 mice developed the highest level of hydrosalpinx, while the susceptible CC042 mice were fully resistant to hydrosalpinx induction. None of the CC mice developed spontaneous hydrosalpinx in the absence of chlamydial infection. The above results, validated macroscopically and microscopically, indicate no correlation between pathology and genital infection, as observed in other inbred mice. Nevertheless, among the two infection-resistant strains, CC012 developed more severe hydrosalpinx than CC011, and hydrosalpinx was positively correlated with live organism shedding from rectal swabs but not from vaginal swabs, supporting a potential role of gastrointestinal chlamydia in chlamydial genital pathogenicity. The above results lay a foundation for using CC mice to further map the genetic determinants that regulate host susceptibility to chlamydial infection and pathogenicity in the female genital tract.

The unique chlamydial developmental cycle comprises three stages: primary differentiation of infectious elementary bodies (EBs) into reticulate bodies (RBs), RB replication, and secondary differentiation into progeny EBs. Extensive chromosome remodeling during RB-to-EB differentiation is thought to be mediated by the histones HctA and HctB. Here, we used an inducible CRISPR interference system to repress hctA, hctB , or both genes during development in Chlamydia trachomatis . Surprisingly, repression of either histone gene alone or in combination caused only modest reductions in EB yield and did not prevent nucleoid condensation during the parental developmental cycle. In contrast, when progeny EBs generated under histone-repressing conditions were used to initiate secondary infections in the absence of inducer, histone deficiency during EB maturation profoundly impaired fitness in the next infection cycle. Secondary cultures initiated with HctA-deficient EBs exhibited a delayed onset of genome replication, consistent with inefficient primary EB-to-RB differentiation, whereas combined repression of hctA and hctB caused both delayed genome replication and persistently reduced genome accumulation, indicative of defects in RB formation and subsequent growth. Repression of hctB alone did not measurably affect genome replication in secondary cultures. Together, these findings reveal a transgenerational role for chlamydial histones and establish chromosome organization during EB maturation as a key determinant of developmental fitness across infection cycles. IMPORTANCE: Chlamydial histones HctA and HctB are unusual among bacterial chromatin-binding proteins in that they share sequence homology with mammalian histones and are developmentally regulated during the formation of infectious particles. Here, we show that reduced expression of HctA and HctB has only modest effects on genome condensation and EB production, consistent with partial functional redundancy between the two histones and suggesting that additional chromatin factors contribute to EB chromosome compaction. In contrast, deficiency of HctA and HctB during EB maturation has profound consequences in the next infection cycle, impairing primary EB-to-RB differentiation and subsequent RB growth. These findings reveal a previously unrecognized transgenerational role for chlamydial histones and establish chromosome organization during EB maturation as a key determinant of developmental fitness across infection cycles.

Chlamydia trachomatis is a leading cause of urogenital infections that can result in serious long-term complications. This obligate intracellular bacterium undergoes a biphasic developmental cycle alternating between the infectious elementary body and the replicative reticulate body and can enter a persistent state in response to adverse environmental conditions. Although transcriptomic reprogramming is central to chlamydial stress adaptation and persistence, how responses differ across biologically distinct stressors remains incompletely defined. Here, we performed a comparative reanalysis of five published, high-quality C. trachomatis RNA-Seq datasets generated under prolonged interferon-γ treatment, tryptophan starvation, iron starvation, penicillin exposure, or acute heat shock. Global transcriptomic analyses reveal extensive stress-specific reprogramming and a clear separation between the transcriptome induced by heat shock and those induced by chronic stresses. Transcriptomic overlap observed among chronic stress conditions is substantially reduced when the heat shock transcriptome is included, indicating that shared transcriptional features are stressor-dependent. Unexpectedly, tryptophan starvation and iron starvation exhibit particularly close transcriptomic similarity, consistent with regulatory cross-talk mediated by the iron-dependent transcriptional repressor YtgR. In contrast, interferon-γ induces a distinct but partially overlapping transcriptome, likely reflecting activation of additional host-mediated antimicrobial mechanisms beyond tryptophan deprivation. Together, these findings demonstrate that adaptation to different biological stressors in C. trachomatis is driven by distinct transcriptomic reprogramming, while consistently involving a subset of functions that may represent points of vulnerability for disrupting chlamydial persistence.