Neisseria gonorrhoeae drives Chlamydia trachomatis into a persistence-like state during in vitro co-infection.

Chlamydia trachomatis and Neisseria gonorrhoeae are the most prevalent bacterial sexually transmitted infections (STIs) globally. Despite frequent co-infections in patients, few studies have investigated how mono-infections may differ from co-infections. We hypothesized that a symbiotic relationship between the pathogens could account for the high rates of clinical co-infection. During in vitro co-infection, we observed an unexpected phenotype where the C. trachomatis developmental cycle was impaired by N. gonorrhoeae. C. trachomatis is an obligate intracellular pathogen with a unique biphasic developmental cycle progressing from infectious elementary bodies (EB) to replicative reticulate bodies (RB), and back. After 12 hours of co-infection, we observed fewer EBs than in a mono-infection. Chlamydial genome copy number remained equivalent between mono- and co-infections. This is a hallmark of Chlamydial persistence. Chlamydial persistence alters inclusion morphology but varies depending on the stimulus/stress. We observed larger, but fewer, Chlamydia during co-infection. Tryptophan depletion can induce Chlamydial persistence, but tryptophan supplementation did not reverse the co-infection phenotype. Only viable and actively growing N. gonorrhoeae produced the inhibition phenotype in C. trachomatis. Piliated N. gonorrhoeae had the strongest effect on C. trachomatis, but hyperpiliated or non-piliated N. gonorrhoeae still produced the phenotype. EB development was modestly impaired when N. gonorrhoeae were grown in transwells above the infected monolayer. C. trachomatis serovar L2 was not impaired during co-infection. Chlamydial impairment could be due to cytoskeletal or osmotic stress caused by an as-yet-undefined mechanism. We conclude that N. gonorrhoeae induces a persistence-like state in C. trachomatis that is serovar dependent.

Retrospective Analysis of Wastewater-Based Epidemiology of SARS-CoV-2 in Residences on a Large College Campus: Relationships between Wastewater Outcomes and COVID-19 Cases across Two Semesters with Different COVID-19 Mitigation Policies.

Wastewater-based epidemiology (WBE) has been utilized for outbreak monitoring and response efforts in university settings during the coronavirus disease 2019 (COVID-19) pandemic. However, few studies examined the impact of university policies on the effectiveness of WBE to identify cases and mitigate transmission. The objective of this study was to retrospectively assess relationships between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wastewater outcomes and COVID-19 cases in residential buildings of a large university campus across two academic semesters (August 2020-May 2021) under different COVID-19 mitigation policies. Clinical case surveillance data of student residents were obtained from the university COVID-19 response program. We collected and processed building-level wastewater for detection and quantification of SARS-CoV-2 RNA by RT-qPCR. The odds of obtaining a positive wastewater sample increased with COVID-19 clinical cases in the fall semester (OR = 1.50, P value = 0.02), with higher odds in the spring semester (OR = 2.63, P value < 0.0001). We observed linear associations between SARS-CoV-2 wastewater concentrations and COVID-19 clinical cases (parameter estimate = 1.2, P value = 0.006). Our study demonstrated the effectiveness of WBE in the university setting, though it may be limited under different COVID-19 mitigation policies. As a complementary surveillance tool, WBE should be accompanied by robust administrative and clinical testing efforts for the COVID-19 pandemic response.

A multistate assessment of population normalization factors for wastewater-based epidemiology of COVID-19.

Wastewater-based epidemiology (WBE) has become a valuable tool for monitoring SARS-CoV-2 infection trends throughout the COVID-19 pandemic. Population biomarkers that measure the relative human fecal contribution to normalize SARS-CoV-2 wastewater concentrations are needed for improved analysis and interpretation of community infection trends. The Centers for Disease Control and Prevention National Wastewater Surveillance System (CDC NWSS) recommends using the wastewater flow rate or human fecal indicators as population normalization factors. However, there is no consensus on which normalization factor performs best. In this study, we provided the first multistate assessment of the effects of flow rate and human fecal indicators (crAssphage, F+ Coliphage, and PMMoV) on the correlation of SARS-CoV-2 wastewater concentrations and COVID-19 cases using the CDC NWSS dataset of 182 communities across six U.S. states. Flow normalized SARS-CoV-2 wastewater concentrations produced the strongest correlation with COVID-19 cases. The correlation from the three human fecal indicators were significantly lower than flow rate. Additionally, using reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) significantly improved correlation values over samples that were analyzed with real-time reverse transcription quantitative polymerase chain reaction (rRT-qPCR). Our assessment shows that utilizing flow normalization with RT-ddPCR generate the strongest correlation between SARS-CoV-2 wastewater concentrations and COVID-19 cases.

The infected and the affected: A longitudinal study of the impact of the COVID-19 pandemic on schoolchildren in Florida.

Objectives: To identify risk factors associated with symptoms of anxiety, depression, and obsessive-compulsive disorder (OCD) among children during the 1st year of the COVID-19 pandemic. Methods: A longitudinal study with three cross-sectional timepoints [April 2020 (n = 273), October 2020 (n = 180), and April 2021 (n = 116)] was conducted at a K-12 public school in Florida. Infection and sero-positivity for SARS-CoV-2 was determined by molecular and serologic approaches. Adjusted odds ratios using mixed effect logistic regression models for symptom-derived indicators of anxiety, depression, and OCD in children in April 2021 are presented; past infection and seropositivity were included in the models. Results: The prevalence of anxiety, depression, or OCD moved from 47.1, to 57.2, to 42.2% across the three timepoints during the study. By endline of the study, in April 2021, non-white children were at higher risk for depression and OCD. Risk for anxiety, depression, and OCD was associated with students who lost a family member due to COVID-19 and who were identified as at-risk in previous timepoints. Rates of SARS-CoV-2 infection and seropositivity were low and not statistically associated with assessed outcomes. Conclusions: In situations like the COVID-19 pandemic, targeted mental health interventions and screenings are needed in children and adolescents, especially among minority children.

Assessment of a mass balance equation for estimating community-level prevalence of COVID-19 using wastewater-based epidemiology in a mid-sized city.

Wastewater-based epidemiology (WBE) has emerged as a valuable epidemiologic tool to detect the presence of pathogens and track disease trends within a community. WBE overcomes some limitations of traditional clinical disease surveillance as it uses pooled samples from the entire community, irrespective of health-seeking behaviors and symptomatic status of infected individuals. WBE has the potential to estimate the number of infections within a community by using a mass balance equation, however, it has yet to be assessed for accuracy. We hypothesized that the mass balance equation-based approach using measured SARS-CoV-2 wastewater concentrations can generate accurate prevalence estimates of COVID-19 within a community. This study encompassed wastewater sampling over a 53-week period during the COVID-19 pandemic in Gainesville, Florida, to assess the ability of the mass balance equation to generate accurate COVID-19 prevalence estimates. The SARS-CoV-2 wastewater concentration showed a significant linear association (Parameter estimate = 39.43, P value < 0.0001) with clinically reported COVID-19 cases. Overall, the mass balance equation produced accurate COVID-19 prevalence estimates with a median absolute error of 1.28%, as compared to the clinical reference group. Therefore, the mass balance equation applied to WBE is an effective tool for generating accurate community-level prevalence estimates of COVID-19 to improve community surveillance.

Erratum: A CTS Team Approach to Wastewater-Based Epidemiology of Non-Typhoidal Salmonella in Gainesville, FL - ERRATUM.

[This corrects the article DOI: 10.1017/cts.2022.23.].

Wastewater surveillance for SARS-CoV-2 in a small coastal community: Effects of tourism on viral presence and variant identification among low prevalence populations.

Wastewater-based epidemiology has been used to measure SARS-CoV-2 prevalence in cities worldwide as an indicator of community health, however, few longitudinal studies have followed SARS-CoV-2 in wastewater in small communities from the start of the pandemic or evaluated the influence of tourism on viral loads. Therefore the objective of this study was to use measurements of SARS-CoV-2 in wastewater to monitor viral trends and variants in a small island community over a twelve-month period beginning May 1, 2020, before the community re-opened to tourists. Wastewater samples were collected weekly and analyzed to detect and quantify SARS-CoV-2 genome copies. Sanger sequencing was used to determine genome sequences from total RNA extracted from wastewater samples positive for SARS-CoV-2. Visitor data was collected from the local Chamber of Commerce. We performed Poisson and linear regression to determine if visitors to the Cedar Key Chamber of Commerce were positively associated with SARS-CoV-2-positive wastewater samples and the concentration of SARS-CoV-2 RNA. Results indicated that weekly wastewater samples were negative for SARS-CoV-2 until mid-July when positive samples were recorded in four of five consecutive weeks. Additional positive results were recorded in November and December 2020, as well as January, March, and April 2021. Tourism data revealed that the SARS-CoV-2 RNA concentration in wastewater increased by 1.06 Log10 genomic copies/L per 100 tourists weekly. Sequencing from six positive wastewater samples yielded two complete sequences of SARS-CoV-2, two overlapping sequences, and two low yield sequences. They show arrival of a new variant SARS-CoV-2 in January 2021. Our results demonstrate the utility of wastewater surveillance for SARS-CoV-2 in a small community. Wastewater surveillance and viral genome sequencing suggest that population mobility likely plays an important role in the introduction and circulation of SARS-CoV-2 variants among communities experiencing high tourism and who have a small population size.

What Is Motion? Recent Advances in the Study of Molecular Movement Patterns of the Peptidoglycan Synthesis Machines.

How proteins move through space and time is a fundamental question in biology. While great strides have been made toward a mechanistic understanding of protein movement, many questions remain. We discuss the biological implications of motion in the context of the peptidoglycan (PG) synthesis machines. We reviewed systems in several bacteria, including Escherichia coli, Bacillus subtilis, and Streptococcus pneumoniae, and present a comprehensive view of our current knowledge regarding movement dynamics. Discrepancies are also addressed because "one size does not fit all". For bacteria to divide, new PG is synthesized and incorporated into the growing cell wall by complex multiprotein nanomachines consisting of PG synthases (transglycosylases [TG] and/or transpeptidases [TP]) as well as a variety of regulators and cytoskeletal factors. Advances in imaging capabilities and labeling methods have revealed that these machines are not static but rather circumferentially transit the cell via directed motion perpendicular to the long axis of model rod-shaped bacteria such as E. coli and B. subtilis. The enzymatic activity of the TG:TPs drives motion in some species while motion is mediated by FtsZ treadmilling in others. In addition, both directed and diffusive motion of the PG synthases have been observed using single-particle tracking technology. Here, we examined the biological role of diffusion regarding transit. Lastly, findings regarding the monofunctional transglycosylases (RodA and FtsW) as well as the Class A PG synthases are discussed. This minireview serves to showcase recent advances, broach mechanistic unknowns, and stimulate future areas of study.

Psychosocial Health of K-12 Students Engaged in Emergency Remote Education and In-Person Schooling: A Cross-Sectional Study.

As online classes became the norm in many countries as a response to the COVID-19 pandemic, the concern for child and adolescent mental health became an issue of concern. This study evaluates the differences in the psychosocial status of school children based on engagement in in-person or Emergency Remote Education (ERE) and assessed the prevalence and predictors of symptom-derived risk levels for anxiety, depression, and obsessive-compulsive disorders (OCD). Cross-sectional data were collected from students at a Florida K-12 school and their household members through an online survey conducted in October 2020 (n = 145). No significant difference was found between ERE and in-person learning for risk of anxiety, depression, or OCD. Prevalence of students presenting as at risk for anxiety, depression, and OCD was 42.1%, 44.8%, and 41.4%. Several student factors (e.g., child sex, school level) and parental factors (e.g., parental COVID-19 attitudes) were associated with students presenting as at risk for anxiety, depression, or OCD; child's participation in sports was protective against all three outcomes. Participation in sports was found to be protective against risk of anxiety (aOR = 0.36, CI = 0.14-0.93), depression (aOR = 0.38, CI = 0.15-0.93), and OCD (aOR = 0.31, CI = 0.11-0.85).

Psychosocial health of school-aged children during the initial COVID-19 safer-at-home school mandates in Florida: a cross-sectional study.

BACKGROUND: Given the emerging literature regarding the impacts of lockdown measures on mental health, this study aims to describe the psychosocial health of school-aged children and adolescents during the COVID-19 Safer-at-Home School mandates. METHODS: A cross-sectional study was conducted in April 2020 (n = 280) among K-12 students at a research school in North Central Florida. Bivariate analysis and logistic and multinomial logistic regression models were used to examine socio-demographic and knowledge, attitude, and practice (KAP) predictors of indicators of anxiety-related, depressive, and obsessive-compulsive disorder(OCD)-related symptoms. Outcomes (anxiety, OCD, and depressive related symptoms) were measured by indices generated based on reported symptoms associated with each psychosocial outcome. RESULTS: Loss of household income was associated with increased risk for all three index-based outcomes: depressive symptoms [aOR = 3.130, 95% CI = (1.41-6.97)], anxiety-related symptoms [aOR = 2.531, 95%CI = (1.154-5.551)], and OCD-related symptoms [aOR = 2.90, 95%CI = (1.32-6.36)]. Being female was associated with being at higher risk for depressive symptoms [aOR = 1.72, 95% CI = (1.02-2.93)], anxiety-related symptoms [aOR = 1.75, 95% CI = (1.04-2.97)], and OCD-related symptoms [aOR = 1.764, 95%CI = (1.027-3.028)]. Parental practices protective against COVID-19 were associated with children being at higher risk of depressive symptoms [aOR = 1.55, 95% CI = (1.04-2.31)]. Lower school level was associated with children being at higher risk of anxiety-related and OCD-related symptoms. CONCLUSIONS: As the COVID-19 pandemic continues, schools should prioritize mental health interventions that target younger, female students, and children of families with income loss. Limiting the spread of COVID-19 through school closure may exacerbate negative psychosocial health outcomes in children, thus school administrators should move quickly to target those at greatest risk.

Competing Substrates for the Bifunctional Diaminopimelic Acid Epimerase/Glutamate Racemase Modulate Peptidoglycan Synthesis in Chlamydia trachomatis.

The Chlamydia trachomatis genome encodes multiple bifunctional enzymes, such as DapF, which is capable of both diaminopimelic acid (DAP) epimerase and glutamate racemase activity. Our previous work demonstrated the bifunctional activity of chlamydial DapF in vitro and in a heterologous system (Escherichia coli). In the present study, we employed a substrate competition strategy to demonstrate DapF Ct function in vivo in C. trachomatis We reasoned that, because DapF Ct utilizes a shared substrate-binding site for both racemase and epimerase activities, only one activity can occur at a time. Therefore, an excess of one substrate relative to another must determine which activity is favored. We show that the addition of excess l-glutamate or meso-DAP (mDAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess l-glutamate reduced in vivo synthesis of mDAP by C. trachomatis to undetectable levels, thus confirming that excess racemase substrate led to inhibition of DapF Ct DAP epimerase activity. We previously showed that expression of dapFCt in a murI (racemase) ΔdapF (epimerase) double mutant of E. coli rescues the d-glutamate auxotrophic defect. Addition of excess mDAP inhibited growth of this strain, but overexpression of dapFCt allowed the mutant to overcome growth inhibition. These results confirm that DapF Ct is the primary target of these mDAP and l-glutamate treatments. Our findings demonstrate that suppression of either the glutamate racemase or epimerase activity of DapF compromises the growth of C. trachomatis Thus, a substrate competition strategy can be a useful tool for in vivo validation of an essential bifunctional enzyme.

Chlamydial MreB Directs Cell Division and Peptidoglycan Synthesis in Escherichia coli in the Absence of FtsZ Activity.

Cell division is the ultimate process for the propagation of bacteria, and FtsZ is an essential protein used by nearly all bacteria for this function. Chlamydiae belong to a small group of bacteria that lack the universal cell division protein FtsZ but still divide by binary fission. Chlamydial MreB is a member of the shape-determining MreB/Mbl family of proteins responsible for rod shape morphology in Escherichia coliChlamydia also encodes a homolog of RodZ, an MreB assembly cytoskeletal protein that links MreB to cell wall synthesis proteins. We hypothesized that MreB directs cell division in Chlamydia and that chlamydial MreB could replace FtsZ function for cell division in E. coli Overexpression of chlamydial mreB-rodZ in E. coli induced prominent morphological changes with production of large swollen or oval bacteria, eventually resulting in bacterial lysis. Low-level expression of chlamydial mreB-rodZ restored viability of a lethal ΔmreB mutation in E. coli, although the bacteria lost their typical rod shape and grew as rounded cells. When FtsZ activity was inhibited by overexpression of SulA in the ΔmreB mutant of E. coli complemented with chlamydial mreB-rodZ, spherical E. coli grew and divided. Localization studies using a fluorescent fusion chlamydial MreB protein indicated that chlamydial RodZ directs chlamydial MreB to the E. coli division septum. These results demonstrate that chlamydial MreB, in partnership with chlamydial RodZ, acts as a cell division protein. Our findings suggest that an mreB-rodZ-based mechanism allows Chlamydia to divide without the universal division protein FtsZ.IMPORTANCE The study of Chlamydia growth and cell division is complicated by its obligate intracellular nature and biphasic lifestyle. Chlamydia also lacks the universal division protein FtsZ. We employed the cell division system of Escherichia coli as a surrogate to identify chlamydial cell division proteins. We demonstrate that chlamydial MreB, together with chlamydial RodZ, forms a cell division and growth complex that can replace FtsZ activity and support cell division in E. coli Chlamydial RodZ plays a major role in directing chlamydial MreB localization to the cell division site. It is likely that the evolution of chlamydial MreB and RodZ to form a functional cell division complex allowed Chlamydia to dispense with its FtsZ-based cell division machinery during genome reduction. Thus, MreB-RodZ represents a possible mechanism for cell division in other bacteria lacking FtsZ.

Chlamydia trachomatis Oligopeptide Transporter Performs Dual Functions of Oligopeptide Transport and Peptidoglycan Recycling.

Peptidoglycan, the sugar-amino acid polymer that composes the bacterial cell wall, requires a significant expenditure of energy to synthesize and is highly immunogenic. To minimize the loss of an energetically expensive metabolite and avoid host detection, bacteria often recycle their peptidoglycan, transporting its components back into the cytoplasm, where they can be used for subsequent rounds of new synthesis. The peptidoglycan-recycling substrate binding protein (SBP) MppA, which is responsible for recycling peptidoglycan fragments in Escherichia coli, has not been annotated for most intracellular pathogens. One such pathogen, Chlamydia trachomatis, has a limited capacity to synthesize amino acids de novo and therefore must obtain oligopeptides from its host cell for growth. Bioinformatics analysis suggests that the putative C. trachomatis oligopeptide transporter OppABCDF (OppABCDF Ct ) encodes multiple SBPs (OppA1 Ct , OppA2 Ct , and OppA3 Ct ). Intracellular pathogens often encode multiple SBPs, while only one, OppA, is encoded in the E. coliopp operon. We hypothesized that the putative OppABCDF transporter of C. trachomatis functions in both oligopeptide transport and peptidoglycan recycling. We coexpressed the putative SBP genes (oppA1Ct , oppA2Ct , oppA3Ct ) along with oppBCDFCt in an E. coli mutant lacking the Opp transporter and determined that all three chlamydial OppA subunits supported oligopeptide transport. We also demonstrated the in vivo functionality of the chlamydial Opp transporter in C. trachomatis Importantly, we found that one chlamydial SBP, OppA3 Ct , possessed dual substrate recognition properties and is capable of transporting peptidoglycan fragments (tri-diaminopimelic acid) in E. coli and in C. trachomatis These findings suggest that Chlamydia evolved an oligopeptide transporter to facilitate the acquisition of oligopeptides for growth while simultaneously reducing the accumulation of immunostimulatory peptidoglycan fragments in the host cell cytosol. The latter property reflects bacterial pathoadaptation that dampens the host innate immune response to Chlamydia infection.

Fosmidomycin, an inhibitor of isoprenoid synthesis, induces persistence in Chlamydia by inhibiting peptidoglycan assembly.

The antibiotic, fosmidomycin (FSM) targets the methylerythritol phosphate (MEP) pathway of isoprenoid synthesis by inhibiting the essential enzyme, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) and is lethal to intracellular parasites and bacteria. The obligate intracellular bacterial pathogen, Chlamydia trachomatis, alternates between two developmental forms: the extracellular, infectious elementary body (EB), and the intracellular, replicative form called the reticulate body (RB). Several stressful growth conditions including iron deprivation halt chlamydial cell division and cause development of a morphologically enlarged, but viable form termed an aberrant body (AB). This phenotype constitutes the chlamydial developmental state known as persistence. This state is reversible as removal of the stressor allows the chlamydiae to re-enter and complete the normal developmental cycle. Bioinformatic analysis indicates that C. trachomatis encodes a homolog of Dxr, but its function and the requirement for isoprenoid synthesis in chlamydial development is not fully understood. We hypothesized that chlamydial Dxr (DxrCT) is functional and that the methylerythritol phosphate (MEP) pathway is required for normal chlamydial development. Thus, FSM exposure should be lethal to C. trachomatis. Overexpression of chlamydial Dxr (DxrCT) in Escherichia coli under FSM exposure and in a conditionally lethal dxr mutant demonstrated that DxrCT functions similarly to E. coli Dxr. When Chlamydia-infected cultures were exposed to FSM, EB production was significantly reduced. However, titer recovery assays, electron microscopy, and peptidoglycan labeling revealed that FSM inhibition of isoprenoid synthesis is not lethal to C. trachomatis, but instead induces persistence. Bactoprenol is a critical isoprenoid required for peptidoglycan precursor assembly. We therefore conclude that FSM induces persistence in Chlamydia by preventing bactoprenol production necessary for peptidoglycan precursor assembly and subsequent cell division.

Distribution and characterization of Shiga toxin converting temperate phages carried by Shigella flexneri in Hispaniola.

Shigella infections account for a considerable burden of acute diarrheal diseases worldwide and remain a major cause of childhood mortality in developing countries. Although, all four species of Shigella (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) cause bacillary dysentery, historically only S. dysenteriae type 1 has been recognized as carrying the genes for Shiga toxin (stx). Recent epidemiological data, however, have suggested that the emergence of stx carrying S. flexneri strains may have originated from bacteriophage-mediated inter-species horizontal gene transfer in one specific geographical area, Hispaniola. To test this hypothesis, we analyzed whole genome sequences of stx-encoding phages carried by S. flexneri strains isolated in Haiti and S. flexneri S. boydii and S. dysenteriae strains isolated from international travelers who likely acquired the infection in Haiti or the Dominican Republic. Phylogenetic analysis showed that phage sequences encoded in the Shigella strains from Hispaniola were bacteriophage φPOC-J13 and they were all closely related to a phage isolated from a USA isolate, E. coli 2009C-3133 serotype O119:H4. In addition, despite the low genetic heterogeneity of phages from different Shigella spp. circulating in the Caribbean island between 2001 and 2014, two distinct clusters emerged in Haiti and the Dominican Republic. Each cluster possibly originated from phages isolated from S. flexneri 2a, and within each cluster several instances of horizontal phage transfer from S. flexneri 2a to other species were detected. The implications of the emergence of stx-producing non-S. dysenteriae type 1 Shigella species, such as S. flexneri, spans not only the basic science behind horizontal phage spread, but also extends to medical treatment of patients infected with this pathogen.

Chlamydia trachomatis dapF Encodes a Bifunctional Enzyme Capable of Both d-Glutamate Racemase and Diaminopimelate Epimerase Activities.

Peptidoglycan is a sugar/amino acid polymer unique to bacteria and essential for division and cell shape maintenance. The d-amino acids that make up its cross-linked stem peptides are not abundant in nature and must be synthesized by bacteria de novo d-Glutamate is present at the second position of the pentapeptide stem and is strictly conserved in all bacterial species. In Gram-negative bacteria, d-glutamate is generated via the racemization of l-glutamate by glutamate racemase (MurI). Chlamydia trachomatis is the leading cause of infectious blindness and sexually transmitted bacterial infections worldwide. While its genome encodes a majority of the enzymes involved in peptidoglycan synthesis, no murI homologue has ever been annotated. Recent studies have revealed the presence of peptidoglycan in C. trachomatis and confirmed that its pentapeptide includes d-glutamate. In this study, we show that C. trachomatis synthesizes d-glutamate by utilizing a novel, bifunctional homologue of diaminopimelate epimerase (DapF). DapF catalyzes the final step in the synthesis of meso-diaminopimelate, another amino acid unique to peptidoglycan. Genetic complementation of an Escherichia coli murI mutant demonstrated that Chlamydia DapF can generate d-glutamate. Biochemical analysis showed robust activity, but unlike canonical glutamate racemases, activity was dependent on the cofactor pyridoxal phosphate. Genetic complementation, enzymatic characterization, and bioinformatic analyses indicate that chlamydial DapF shares characteristics with other promiscuous/primordial enzymes, presenting a potential mechanism for d-glutamate synthesis not only in Chlamydia but also numerous other genera within the Planctomycetes-Verrucomicrobiae-Chlamydiae superphylum that lack recognized glutamate racemases.IMPORTANCE Here we describe one of the last remaining "missing" steps in peptidoglycan synthesis in pathogenic Chlamydia species, the synthesis of d-glutamate. We have determined that the diaminopimelate epimerase (DapF) encoded by Chlamydia trachomatis is capable of carrying out both the epimerization of DAP and the pyridoxal phosphate-dependent racemization of glutamate. Enzyme promiscuity is thought to be the hallmark of early microbial life on this planet, and there is currently an active debate as to whether "moonlighting enzymes" represent primordial evolutionary relics or are a product of more recent reductionist evolutionary pressures. Given the large number of Chlamydia species (as well as members of the Planctomycetes-Verrucomicrobiae-Chlamydiae superphylum) that possess DapF but lack homologues of MurI, it is likely that DapF is a primordial isomerase that functions as both racemase and epimerase in these organisms, suggesting that specialized d-glutamate racemase enzymes never evolved in these microbes.

A Brief History of Shigella.

The history of Shigella, the causative agent of bacillary dysentery, is a long and fascinating one. This brief historical account starts with descriptions of the disease and its impact on human health from ancient time to the present. Our story of the bacterium starts just before the identification of the dysentery bacillus by Kiyoshi Shiga in 1898 and follows the scientific discoveries and principal scientists who contributed to the elucidation of Shigella pathogenesis in the first 100 years. Over the past century, Shigella has proved to be an outstanding model of an invasive bacterial pathogen and has served as a paradigm for the study of other bacterial pathogens. In addition to invasion of epithelial cells, some of those shared virulence traits include toxin production, multiple-antibiotic resistance, virulence genes encoded on plasmids and bacteriophages, global regulation of virulence genes, pathogenicity islands, intracellular motility, remodeling of host cytoskeleton, inflammation/polymorphonuclear leukocyte signaling, apoptosis induction/inhibition, and "black holes" and antivirulence genes. While there is still much to learn from studying Shigella pathogenesis, what we have learned so far has also contributed greatly to our broader understanding of bacterial pathogenesis.

Investigating the Relatedness of Enteroinvasive Escherichia coli to Other E. coli and Shigella Isolates by Using Comparative Genomics.

Enteroinvasive Escherichia coli (EIEC) is a unique pathovar that has a pathogenic mechanism nearly indistinguishable from that of Shigella species. In contrast to isolates of the four Shigella species, which are widespread and can be frequent causes of human illness, EIEC causes far fewer reported illnesses each year. In this study, we analyzed the genome sequences of 20 EIEC isolates, including 14 first described in this study. Phylogenomic analysis of the EIEC genomes demonstrated that 17 of the isolates are present in three distinct lineages that contained only EIEC genomes, compared to reference genomes from each of the E. coli pathovars and Shigella species. Comparative genomic analysis identified genes that were unique to each of the three identified EIEC lineages. While many of the EIEC lineage-specific genes have unknown functions, those with predicted functions included a colicin and putative proteins involved in transcriptional regulation or carbohydrate metabolism. In silico detection of the Shigella virulence plasmid (pINV), which is essential for the invasion of host cells, demonstrated that a form of pINV was present in nearly all EIEC genomes, but the Mxi-Spa-Ipa region of the plasmid that encodes the invasion-associated proteins was absent from several of the EIEC isolates. The comparative genomic findings in this study support the hypothesis that multiple EIEC lineages have evolved independently from multiple distinct lineages of E. coli via the acquisition of the Shigella virulence plasmid and, in some cases, the Shigella pathogenicity islands.

Pathogenic Chlamydia Lack a Classical Sacculus but Synthesize a Narrow, Mid-cell Peptidoglycan Ring, Regulated by MreB, for Cell Division.

The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe's developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.

Evolution of Bacterial Pathogens Within the Human Host.

Selective pressures within the human host, including interactions with innate and adaptive immune responses, exposure to medical interventions such as antibiotics, and competition with commensal microbiota all facilitate the evolution of bacterial pathogens. In this chapter, we present examples of pathogen strategies that emerged as a result of selective pressures within the human host niche and discuss the resulting coevolutionary "arms race" between these organisms. In bacterial pathogens, many of the genes responsible for these strategies are encoded on mobile pathogenicity islands or plasmids, underscoring the importance of horizontal gene transfer in the emergence of virulent microbial species.