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The Cluster Validity Index is an integral part of clustering algorithms. It evaluates inter-cluster separation and intra-cluster cohesion of candidate clusters to determine the quality of potential solutions. Several cluster validity indices have been suggested for both classical clustering algorithms and automatic metaheuristic-based clustering algorithms. Different cluster validity indices exhibit different characteristics based on the mathematical models they employ in determining the values for the various cluster attributes. Metaheuristic-based automatic clustering algorithms use cluster validity index as a fitness function in its optimization procedure to evaluate the candidate cluster solution's quality. A systematic review of the cluster validity indices used as fitness functions in metaheuristic-based automatic clustering algorithms is presented in this study. Identifying, reporting, and analysing various cluster validity indices is important in classifying the best CVIs for optimum performance of a metaheuristic-based automatic clustering algorithm. This review also includes an experimental study on the performance of some common cluster validity indices on some synthetic datasets with varied characteristics as well as real-life datasets using the SOSK-means automatic clustering algorithm. This review aims to assist researchers in identifying and selecting the most suitable cluster validity indices (CVIs) for their specific application areas., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2025 The Authors.)

Background: Malaria and anemia are significant public health concerns that contribute to child mortality in African. Despite global efforts to control the two diseases, their prevalence in high-risk regions like Nigeria remains high. Understanding socioeconomic, demographic, and geographical factors associated with malaria and anemia, is critical for effective intervention strategies., Objective: The study aims to assess the risk factors for malaria and anemia in children under 5 years using the joint generalized linear mixed model (JGLMM)., Methods: The Nigeria Malaria Indicator Survey (NMIS) 2021 dataset was used, with a sample of 10,120 children aged 6-59 months. A two-stage sampling method was applied. Descriptive statistics and chi-square tests examined factors associated with malaria and anemia prevalence. Missing data were handled using multiple imputations with chained equations (MICE). Lastly, the adjusted odds ratio was interpreted for the current study. SAS was used in analyzing the data and statistical significance was set at 5% significance level., Results: The prevalence of malaria and anemia was 36.81 and 67.66%, respectively, in children between 6 and 59 months old in Nigeria. The JGLMM was used to examine malaria RDT and anemia findings in conjunction with demographic, geographic, and socioeconomic covariates. The following underlying risk factors for malaria and anemia in children were discovered in the study: region, altitude, age of child in months, toilet facility of the household, main wall material used for the house, main roof material used for the house, children under five who slept under a mosquito net, whether the child had fever in last 2 weeks before the survey took place, place of residence where the child resides, household wealth index, sex of child, and mother's education level. However, whether the mother knew of ways to prevent malaria was not statistically significant regarding anemia., Conclusion: This study highlights the importance of addressing fever as a key factor for anemia and improving housing conditions to reduce malaria and anemia prevalence. Policymakers should prioritize mosquito net distribution and healthcare access, particularly in rural areas. The study's novelty lies in its handling of missing data through imputation techniques, enhancing the reliability of findings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Mhelembe, Ramroop and Habyarimana.)

Background: Anaemia, malaria, and stunting remain health problems, especially in children younger than five years, and those conditions are linked to morbidity and mortality. The main objective was to assess the relationships between anaemia, malaria, and stunting. Also, the current study aimed to understand the complex interrelationships between explanatory factors, and their direct or indirect relationship with childhood malaria, anaemia, and stunting in Burundi., Methods: The study used secondary data from the Demographic and Health Survey in Burundi (BDHS) conducted on the March 7, 2017, with a weighted sample size of 13611 children younger than five years. A multivariate structural equation model (SEM) was used to evaluate the interrelationships between dependent variables and their direct or indirect relationship with childhood malaria, anaemia, and stunting. SEMs diverge from other techniques, as they look at the effects on hypothesised relationships from both direct and indirect perspectives (Takele et al., 2023) [1]. The variables with statistical significance were set at a p-value <0.05., Results: The findings from this study indicated an association between anaemia, malaria, and stunting (p < 0.001). The environmental and household factors were statistically significant (p < 0.038 and p < 0.001 respectively) and positively impacted childhood malaria, anaemia, and stunting. The results also indicated that the household factors were statistically significant (p < 0.001) predictors of childhood malaria, anaemia, and stunting. Furthermore, the findings from this study revealed that geophysical factors have a positive significant (p < 0.001) impact on childhood malaria, anaemia, and stunting via the mediating of the household factors. Contrastingly, with the environmental factors as a mediator, we observe a negative significant (p < 0.001) impact on childhood malaria, anaemia, and stunting. Lastly, the results showed that demographic factors had a negative significant (p = 0.004) effect on childhood anaemia, malaria, and stunting via the mediating of household factors., Conclusion: The findings from this study revealed an association between malaria, anaemia, and stunting, which imply that these conditions could contribute to collaborative improvements in child well-being. In addition, child demographic, household, environmental, and geographic factors were direct and indirect important drivers of childhood malaria, anaemia, and stunting. Therefore, improving sanitation, access to clean water, nutrition practices, and health care, especially for children from rural areas, and uneducated mothers with poor backgrounds could help to control and eliminate stunting, anemia, and malaria in children younger than five years in Burundi., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background: Malnutrition is one of the most critical health challenges confronting public health agencies in developing nations. This study aimed to determine the scope and underlying factors contributing to malnutrition in West African countries, specifically Gabon, Gambia, Liberia, Mauritania, and Nigeria., Method: For this secondary data analysis, this study drew upon the demographic and health surveys (DHS) conducted within these West African nations. These surveys employed a complex sampling design involving a combination of stratification and cluster sampling in two stages, with varying probabilities of selection leading to weighted samples that effectively represented different components of the population. Given the intricacies of this sampling design, it is paramount to account for them when analyzing the survey data. To address this concern, this study applied a survey logistic regression model, which accommodates factors such as stratification, clustering, and sampling weights and departs from the assumption of independence inherent in the ordinary logistic regression model., Results: The outcomes of this model revealed several variables that emerged as statistically significant (p < 0.05) determinants of malnutrition. These influential factors encompass the region of the respondent, the current age of the mother, the highest level of education attained by the mother, the source of drinking water, the type of toilet facility, the household's wealth status, the age and gender of the child, and whether the child experienced a fever in the preceding two weeks., Conclusion: These findings demonstrate with poignant clarity the importance of primary health care interventions in the recognition and management of malnutrition. The countries of interest should invest in public health care interventions including community workshops and outreach programs. Workshops may occur at primary health care facilities during queue waits, or health workers may work with community leaders to perform workshops in areas of high foot traffic, such as places of worship, shopping hubs and collection points for financial aid or grants., (© 2024. The Author(s).)

Notwithstanding the interventions implemented to address child mortality, anaemia and malnutrition remain a concern for the future of developing countries. Anaemia and malnutrition contribute a high proportion of the causes of childhood morbidity in Africa. The objective of this study is to jointly model anaemia and malnutrition using a copula geo-additive model. This study is a secondary data analysis where a Demographic and Health Survey of 2016 data from Angola, Malawi, and Senegal was used. The descriptive analysis was conducted in SPSS and the copula geo-additive model analysis was performed in R 3.63. The results showed that female children are notably associated with anaemia and a malnourished status (female estimate = 0.144, p -value = 0.027 for anaemia; female estimate = -0.105, p -value = 000 for malnutrition). The probability of each result decreased with an improvement in the mother's level of schooling. This indicates an urgent requirement for interventions to be implemented by policymakers in order to manage children's mortality rates. These interventions can include the introduction of educational programs for older adults, children's dietary programs, and income generation initiatives (starting a small business, etc.). It is hoped that this paper can foster the utilization of copula methodology in this field of science with the use of cross-sectional data.

Background: Anaemia and stunting remain jointly a serious health issue worldwide especially in developing countries. In Lesotho, their prevalence is high, particularly among children less than 5 years of age., Objectives: The primary objective was to determine the association between anaemia and stunting, and identify factors relating to both conditions among children younger than 5 years in Lesotho., Methods: This cross-sectional study used secondary data from 3112 children collected during the 2014 Lesotho Demographic Health Survey (LDHS). Haemoglobin (Hb) levels were adjusted for altitude and a level less than 11 g per deciliters (11 g/dl) was determined as the cutoff for being anaemic. A child with the height-for-age z score (HAZ) below minus two standard deviations (SD) was considered to have stunting. We linked factors relating to anaemia and stunting using a multivariate joint model under the scope of the generalized linear mixed model (GLMM)., Results: The prevalence of anaemia and stunting in children younger than 5 years were 51% and 43% respectively. The multivariate results revealed a strong association between anaemia and stunting. In addition, maternal education, urban vs. rural residence, wealth index and childbirth weight significantly impacted childhood stunting or malnutrition, while having fever and/or diarrhoea was linked to anaemia. Lastly, age was shown to have a significant effect on both stunting and anaemia., Conclusion: Anaemia and stunting or malnutrition showed linked longitudinal trajectories, suggesting both conditions could lead to synergetic improvements in overall child health. Demographic, socio-economic, and geographical characteristics were also important drivers of stunting and anaemia in children younger than 5 years. Thus, children living in similar resources settings as Lesotho could benefit from coordinated programs designed to address both malnutrition and anaemia., (© 2022. The Author(s).)

Background: Malaria and anaemia jointly remain a public health problem in developing countries of which Malawi is one. Although there is an improvement along with intervention strategies in fighting against malaria and anaemia in Malawi, the two diseases remain significant problems, especially in children 6-59 months of age. The main objective of this study was to examine the association between malaria and anaemia. Moreover, the study investigated whether socio-economic, geographic, and demographic factors had a significant impact on malaria and anaemia., Data and Methodology: The present study used a secondary cross-sectional data set from the 2017 Malawi Malaria Indicator Survey (MMIS) with a total number of 2 724 children 6-9 months of age. The study utilized a multivariate joint model within the ambit of the generalized linear mixed model (GLMM) to analyse the data. The two response variables for this study were: the child has either malaria or anaemia., Results: The prevalence of malaria was 37.2% of the total number of children who were tested using an RDT, while 56.9% were anaemic. The results from the multivariate joint model under GLMM indicated a positive association between anaemia and malaria. Furthermore, the same results showed that mother's education level, child's age, the altitude of the place of residence, place of residence, toilet facility, access to electricity and children who slept under a mosquito bed net the night before the survey had a significant effect on malaria and anaemia., Conclusion: The study indicated that there is a strong association between anaemia and malaria. This is interpreted to indicate that controlling for malaria can result in a reduction of anaemia. The socio-economic, geographical and demographic variables have a significant effect on improving malaria and anaemia. Thus, improving health care, toilet facilities, access to electricity, especially in rural areas, educating the mothers of children and increasing mosquito bed nets would contribute in the reduction of malaria and anaemia in Malawi., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors.)

The main objective of this study was to assess the risk factors and spatial correlates of domestic violence against women of reproductive age in Rwanda. A structured spatial approach was used to account for the nonlinear nature of some covariates and the spatial variability on domestic violence. The nonlinear effect was modeled through second-order random walk, and the structured spatial effect was modeled through Gaussian Markov Random Fields specified as an intrinsic conditional autoregressive model. The data from the Rwanda Demographic and Health Survey 2014/2015 were used as an application. The findings of this study revealed that the risk factors of domestic violence against women are the wealth quintile of the household, the size of the household, the husband or partner's age, the husband or partner's level of education, ownership of the house, polygamy, the alcohol consumption status of the husband or partner, the woman's perception of wife-beating attitude, and the use of contraceptive methods. The study also highlighted the significant spatial variation of domestic violence against women at district level.

Malaria is a major public health risk in Rwanda where children and pregnant women are most vulnerable. This infectious disease remains the main cause of morbidity and mortality among children in Rwanda. The main objectives of this study were to assess the prevalence of malaria among children aged six months to 14 years old in Rwanda and to identify the factors associated with malaria in this age group. This study used data from the 2017 Rwanda Malaria Indicator Survey. Due to the complex design used in sampling, a survey logistic regression model was used to fit the data and the outcome variable was the presence or absence of malaria. This study considered 8209 children in the analysis and the prevalence of malaria was 14.0%. This rate was higher among children aged 5-9 years old (15.6%), compared to other age groups. Evidently, the prevalence of malaria was also higher among children from poor families (19.4%) compared to children from the richest families (4.3%). The prevalence of malaria was higher among children from rural households (16.2%) compared to children from urban households (3.4%). The results revealed that other significant factors associated with malaria were: the gender of the child, the number of household members, whether the household had mosquito bed nets for sleeping, whether the dwelling had undergone indoor residual spraying in the 12 months prior to the survey, the location of the household's source of drinking water, the main wall materials of the dwelling, and the age of the head of the household. The prevalence of malaria was also high among children living in houses with walls built from poorly suited materials; this suggests the need for intervention in construction materials. Further, it was found that the Eastern Province also needs special consideration in malaria control due to the higher prevalence of the disease among its residents, compared to those in other provinces.

Anemia among women of child bearing age is a global public health problem. In developing countries such as Rwanda, women and their children may be particularly susceptible to anemia. The main objectives of this study were to assess the prevalence of anemia among women of reproductive age in Rwanda and to identify the risk factors associated therewith. The current study considered socio-economic, demographic and environmental factors and the 2014/2015 Rwanda Demographic and Health Survey data was used for this purpose. Due to the complexity of the sampling design, the present study used survey logistic model that takes account of sampling weight, stratification and clustering. The SAS statistical software was used for the analysis of the results. The anemia status was assessed among 6 680 women aged between 15 and 49 years old and the prevalence of anemia among women of this child bearing age group, was found to be 19.2%. The study also revealed that body mass index, contraceptive use, use of mosquito bed nets, marital status, wealth quintile of the household, size of the household, literacy, tobacco use, type of cooking fuel, type of toilet facilities, type of source of drinkable water, and province of residence, were all factors associated with anemia among women of reproductive age in Rwanda. Hence the current study highlights several health, geographical and socio-economic issues that can assist health care professionals and other relevant authorities in fostering an environment that reduces the risk of anemia for women and children.

Contraceptive use is considered as essential for protecting women's health and rights, influencing fertility and population growth, and helping to promote economic development. The main objective of this study was to analysis the factors and spatial correlates of contraceptive use among women of childbearing age. The 2015 Rwanda Demographic and Health Survey (RDHS) data were used to identify the factors associated with contraceptive use in Rwanda. A Bayesian geo-additive model was used in order to account for fixed effects, nonlinear effects, spatial and random effects inherent in the data. The overall prevalence of use of any contraceptive method among married women of childbearing age in Rwanda was 52.7%. A woman's age, wealth quintile, level of education, working status, number of living children, and exposure to the media was found to increase contraceptive use. The findings from the study also found disparities in contraceptive use at provincial and district level, where prevalence was higher in districts of Northern provinces and lower in districts of western provinces. The findings of this study suggest that exposure to information on contraceptive use in health centres, empowerment of women to access quality contraceptive-use services and religions to play an important role in explaining and informing their adherents on the importance of using a contraceptive method.

Lung endothelial cell apoptosis and injury occur throughout all stages of acute lung injury/acute respiratory distress syndrome and impact disease progression. Caspases 1, 4, and 5 are essential for completion of the apoptotic program known as pyroptosis that also involves proinflammatory cytokines. Because gasdermin D (GSDMD) mediates pyroptotic death and is essential for pore formation, we hypothesized that it might direct caspase 1-encapsulated microparticle (MP) release and mediate endothelial cell death. Our present work provides evidence that GSDMD is released by LPS-stimulated THP-1 monocytic cells, where it is packaged into microparticles together with active caspase 1. Furthermore, only MP released from stimulated monocytic cells that contain both cleaved GSDMD and active caspase 1 induce endothelial cell apoptosis. MPs pretreated with caspase 1 inhibitor Y-VAD or pan-caspase inhibitor Z-VAD do not contain cleaved GSDMD. MPs from caspase 1-knockout cells are also deficient in p30 active GSDMD, further confirming that caspase 1 regulates GSDMD function. Although control MPs contained cleaved GSDMD without caspase 1, these fractions were unable to induce cell death, suggesting that encapsulation of both caspase 1 and GSDMD is essential for cell death induction. Release of microparticulate active caspase 1 was abrogated in GSDMD knockout cells, although cytosolic caspase 1 activation was not impaired. Last, higher concentrations of microparticulate GSDMD were detected in the plasma of septic patients with acute respiratory distress syndrome than in that of healthy donors. Taken together, these findings suggest that GSDMD regulates the release of microparticulate active caspase 1 from monocytes essential for induction of cell death and thereby may play a critical role in sepsis-induced endothelial cell injury.

Childhood anemia is among the most significant health problems faced by public health departments in developing countries. This study aims at assessing the determinants and possible spatial effects associated with childhood anemia in Rwanda. The 2014/2015 Rwanda Demographic and Health Survey (RDHS) data was used. The analysis was done using the structured spatial additive quantile regression model. The findings of this study revealed that the child's age; the duration of breastfeeding; gender of the child; the nutritional status of the child (whether underweight and/or wasting); whether the child had a fever; had a cough in the two weeks prior to the survey or not; whether the child received vitamin A supplementation in the six weeks before the survey or not; the household wealth index; literacy of the mother; mother's anemia status; mother's age at the birth are all significant factors associated with childhood anemia in Rwanda. Furthermore, significant structured spatial location effects on childhood anemia was found.

The continued emergence and spread of infectious agents is of great concern, and systems biology approaches to infectious disease research can advance our understanding of host-pathogen relationships and facilitate the development of new therapies and vaccines. Molecular characterization of infectious samples outside of appropriate biosafety containment can take place only subsequent to pathogen inactivation. Herein, we describe a modified Folch extraction using chloroform/methanol that facilitates the molecular characterization of infectious samples by enabling simultaneous pathogen inactivation and extraction of proteins, metabolites, and lipids for subsequent mass spectrometry-based multi-omics measurements. This single-sample metabolite, protein and lipid extraction (MPLEx) method resulted in complete inactivation of clinically important bacterial and viral pathogens with exposed lipid membranes, including Yersinia pestis, Salmonella Typhimurium, and Campylobacter jejuni in pure culture, and Yersinia pestis, Campylobacter jejuni, and West Nile, MERS-CoV, Ebola, and influenza H7N9 viruses in infection studies. In addition, >99% inactivation, which increased with solvent exposure time, was also observed for pathogens without exposed lipid membranes including community-associated methicillin-resistant Staphylococcus aureus, Clostridium difficile spores and vegetative cells, and adenovirus type 5. The overall pipeline of inactivation and subsequent proteomic, metabolomic, and lipidomic analyses was evaluated using a human epithelial lung cell line infected with wild-type and mutant influenza H7N9 viruses, thereby demonstrating that MPLEx yields biomaterial of sufficient quality for subsequent multi-omics analyses. Based on these experimental results, we believe that MPLEx will facilitate systems biology studies of infectious samples by enabling simultaneous pathogen inactivation and multi-omics measurements from a single specimen with high success for pathogens with exposed lipid membranes.

Salmonella enterica serovar Typhimurium is a food-borne pathogen that causes severe gastroenteritis. The ability of Salmonella to cause disease depends on two type III secretion systems (T3SSs) encoded in two distinct Salmonella pathogenicity islands, 1 and 2 (SPI1 and SPI2, respectively). S. Typhimurium encodes a solo LuxR homolog, SdiA, which can detect the acyl-homoserine lactones (AHLs) produced by other bacteria and upregulate the rck operon and the srgE gene. SrgE is predicted to encode a protein of 488 residues with a coiled-coil domain between residues 345 and 382. In silico studies have provided conflicting predictions as to whether SrgE is a T3SS substrate. Therefore, in this work, we tested the hypothesis that SrgE is a T3SS effector by two methods, a β-lactamase activity assay and a split green fluorescent protein (GFP) complementation assay. SrgE with β-lactamase fused to residue 40, 100, 150, or 300 was indeed expressed and translocated into host cells, but SrgE with β-lactamase fused to residue 400 or 488 was not expressed, suggesting interference by the coiled-coil domain. Similarly, SrgE with GFP S11 fused to residue 300, but not to residue 488, was expressed and translocated into host cells. With both systems, translocation into host cells was dependent upon SPI2. A phylogenetic analysis indicated that srgE is found only within Salmonella enterica subspecies. It is found sporadically within both typhoidal and nontyphoidal serovars, although the SrgE protein sequences found within typhoidal serovars tend to cluster separately from those found in nontyphoidal serovars, suggesting functional diversification., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Yersinia enterocolitica biovar 1B employs two type three secretion systems (T3SS), Ysa and Ysc, which inject effector proteins into macrophages to prevent phagocytosis. Conversely, Salmonella enterica serovar Typhimurium uses a T3SS encoded by Salmonella pathogenicity island 1 (SPI1) to actively invade cells that are normally nonphagocytic and a second T3SS encoded by SPI2 to survive within macrophages. Given the distinctly different outcomes that occur with regard to host cell uptake of S. Typhimurium and Y. enterocolitica, we investigated how each pathogen influences the internalization outcome of the other. Y. enterocolitica reduces S. Typhimurium invasion of HeLa and Caco-2 cells to a level similar to that observed using an S. Typhimurium SPI1 mutant alone. However, Y. enterocolitica had no effect on S. Typhimurium uptake by J774.1 or RAW264.7 macrophage-like cells. Y. enterocolitica was also able to inhibit the invasion of epithelial and macrophage-like cells by Listeria monocytogenes. Y. enterocolitica mutants lacking either the Ysa or Ysc T3SS were partially defective, while double mutants were completely defective, in blocking S. Typhimurium uptake by epithelial cells. S. Typhimurium encodes a LuxR homolog, SdiA, which detects N-acylhomoserine lactones (AHLs) produced by Y. enterocolitica and upregulates the expression of an invasin (Rck) and a putative T3SS effector (SrgE). Two different methods of constitutively activating the S. Typhimurium SdiA regulon failed to reverse the uptake blockade imposed by Y. enterocolitica.

During late stages of infection and prior to lysis of the infected macrophages or amoeba, the Legionella pneumophila-containing phagosome becomes disrupted, followed by bacterial escape into the host cell cytosol, where the last few rounds of bacterial proliferation occur prior to lysis of the plasma membrane. This coincides with growth transition into the post-exponential (PE) phase, which is controlled by regulatory cascades including RpoS and the LetA/S two-component regulator. Whether the temporal expression of flagella by the regulatory cascades at the PE phase is exhibited within the phagosome or after bacterial escape into the host cell cytosol is not known. We have utilized fluorescence microscopy-based phagosome integrity assay to differentiate between vacuolar and cytosolic bacteria/or bacteria within disrupted phagosomes. Our data show that during late stages of infection, expression of FlaA is triggered after bacterial escape into the macrophage cytosol and the peak of FlaA expression is delayed for few hours after cytosolic residence of the bacteria. Importantly, bacterial escape into the host cell cytosol is independent of flagella, RpoS and the two-component regulator LetA/S, which are all triggered by L. pneumophila upon growth transition into the PE phase. Disruption of the phagosome and bacterial escape into the cytosol of macrophages is independent of the bacterial pore-forming activity, and occurs prior to the induction of apoptosis during late stages of infection. We conclude that the temporal and spatial engagement of virulence-associated regulatory cascades by L. pneumophila at the PE phase is temporally and spatially triggered after phagosomal escape and bacterial residence in the host cell cytosol.

Although most Dot/Icm-translocated effectors of Legionella pneumophila are not required for intracellular proliferation, the eukaryotic-like ankyrin effectors, AnkH and AnkJ are required for intracellular proliferation. In this report, we show that the IcmSW chaperones are essential for translocation of AnkJ but not AnkH. The 10 C-terminal residues and the ANK domains of AnkH and AnkJ are required for translocation. Our data indicate that the two ANK domains of AnkH are critical domains required for the function of the effector in intracellular replication of L. pneumophila. The ankH and ankJ mutants are severely defective in intrapulmonary proliferation in mice. Expression of AnkH and AnkJ fusions within HEK293 cells show a punctuate distribution in the cytosol but no association with endocytic vesicles, the Golgi apparatus or the endoplasmic reticulum. Interestingly, the defect in intracellular proliferation of the ankH or ankJ mutants is rescued in HEK293 cells expressing the respective protein. We conclude that AnkH and AnkJ are effectors translocated by the Dot/Icm system by distinct mechanisms and modulate distinct cytosolic processes in the host cell.

The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the (9)L(10)P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-(9)L(10)P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-(9)L(10)P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.

The Dot/Icm type IV secretion system of Legionella pneumophila translocates numerous bacterial effectors into the host cell and is essential for bacterial proliferation within macrophages and protozoa. We have recently shown that L. pneumophila strain AA100/130b harbours 11 genes encoding eukaryotic-like ankyrin (Ank) proteins, a family of proteins involved in various essential eukaryotic cellular processes. In contrast to most Dot/Icm-exported substrates, which have little or no detectable role in intracellular proliferation, a mutation in ankB results in a severe growth defect in intracellular replication within human monocyte-derived macrophages (hMDMs), U937 macrophages and Acanthamoeba polyphaga. Single cell analyses of coinfections of hMDMs have shown that the intracellular growth defect of the ankB mutant is totally rescued in cis within communal phagosomes harbouring the wild type strain. Interestingly, distinct from dot/icm structural mutants, the ankB mutant is also rescued in trans within cells harbouring the wild type strain in a different phagosome, indicating that AnkB is a trans-acting secreted effector. Using adenylate cyclase fusions to AnkB, we show that AnkB is translocated into the host cell via the Dot/Icm secretion system in an IcmSW-dependent manner and that the last three C-terminal amino acid residues are essential for translocation. Distinct from the dot/icm structural mutants, the ankB mutant-containing phagosomes exclude late endosomal and lysosomal markers and their phagosomes are remodelled by the rough endoplasmic reticulum. We show that at the postexponential phase of growth, the LetA/S and PmrA/B Two Component Systems confer a positive regulation on expression of the ankB gene, whereas RpoS, LetE and RelA suppress its expression. Our data show that the eukaryotic-like AnkB protein is a Dot/Icm-exported effector that plays a major role in intracellular replication of L. pneumophila within macrophages and protozoa, and its expression is temporally controlled by regulators of the postexponential phase of growth.

Legionella pneumophila is a ubiquitous organism in the aquatic environment where it is capable of invasion and intracellular proliferation within various protozoan species and is also capable of causing pneumonia in humans. In silico analysis showed that the three sequenced L. pneumophila genomes each contained a common multigene family of 11 ankyrin (ank) genes encoding proteins with approximately 30-35 amino acid tandem Ankyrin repeats that are involved in protein-protein interactions in eukaryotic cells. To examine whether the ank genes are involved in tropism of protozoan hosts, we have constructed isogenic mutants of L. pneumophila in ten of the ank genes. Among the mutants, the DeltaankH and DeltaankJ mutants exhibit significant defects in robust intracellular replication within A. polyphaga, Hartmanella vermiformis and Tetrahymena pyriformis. A similar defect is also exhibited in human macrophages. Most of the ank genes are upregulated by L. pneumophila upon growth transition into the post-exponential phase in vitro and within Acanthamoeba polyphaga, and this upregulation is mediated, at least in part, by RpoS. Single-cell analyses have shown that upon co-infection of the wild-type strain with the ankH or ankJ mutant, the replication defect of the mutant is rescued within communal phagosomes harbouring the wild-type strain, similar to dot/icm mutants. Therefore, at least two of the L. pneumophila eukaryotic-like Ank proteins play a role in intracellular replication of L. pneumophila within amoeba, ciliated protozoa and human macrophages. The Ank proteins may not be involved in host tropism in the aquatic environment. Many of the L. pneumophila eukaryotic-like ank genes are triggered upon growth transition into post-exponential phase in vitro as well as within A. polyphaga. Our data suggest a role for AnkH and AnkJ in modulation of phagosome biogenesis by L. pneumophila independent of evasion of lysosomal fusion and recruitment of the rough endoplasmic reticulum.