Comparison of real-time quantitative PCR and culture for the diagnosis of emerging Rickettsioses

Background

Isolation of Rickettsia species from skin biopsies may be replaced by PCR. We evaluated culture sensitivity compared to PCR based on sampling delay and previous antibiotic treatment.

Methodology/Principal Findings

Skin biopsies and ticks from patients with suspected Rickettsia infection were screened for Rickettsia spp. using qPCR, and positive results were amplified and sequenced for the gltA and ompA genes. Immunofluorescence for spotted fever group rickettsial antigens was done for 79 patients. All skin biopsies and only ticks that tested positive using qPCR were cultured in human embryonic lung (HEL) fibroblasts using the centrifugation-shell vial technique. Patients and ticks were classified as definitely having rickettsioses if there was direct evidence of infection with a Rickettsia sp. using culture or molecular assays or in patients if serology was positive. Data on previous antibiotic treatments were obtained for patients with rickettsiosis. Rickettsia spp. infection was diagnosed in 47 out of 145 patients (32%), 41 by PCR and 12 by culture, whereas 3 isolates were obtained from PCR negative biopsies. For 3 of the patients serology was positive although PCR and culture were negative. Rickettsia africae was the most common detected species (n = 25, [17.2%]) and isolated bacterium (n = 5, [3.4%]). The probability of isolating Rickettsia spp. was 12 times higher in untreated patients and 5.4 times higher in patients from our hometown. Rickettsia spp. was amplified in 24 out of 95 ticks (25%) and we isolated 7 R. slovaca and 1 R. raoultii from Dermacentor marginatus.

Conclusions/Significance

We found a positive correlation between the bacteria copies and the isolation success in skin biopsies and ticks. Culture remains critical for strain analysis but is less sensitive than serology and PCR for the diagnosis of a Rickettsia infection.     

Molecular Detection and Identification of Spotted Fever Group Rickettsiae in Ticks Collected from the West Bank,Palestinian Territories

Background

Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) rickettsiae. Although Spotted Fever is prevalent in the Middle East, no reports for the presence of tick-borne pathogens are available or any studies on the epidemiology of this disease in the West Bank. We aimed to identify the circulating hard tick vectors and genetically characterize SFG Rickettsia species in ixodid ticks from the West Bank-Palestinian territories.

Methodology/Principal Findings

A total of 1,123 ixodid ticks belonging to eight species (Haemaphysalis parva, Haemaphysalis adleri, Rhipicephalus turanicus, Rhipicephalus sanguineus, Rhipicephalus bursa, Hyalomma dromedarii, Hyalomma aegyptium and Hyalomma impeltatum) were collected from goats, sheep, camels, dogs, a wolf, a horse and a tortoise in different localities throughout the West Bank during the period of January-April, 2014. A total of 867 ticks were screened for the presence of rickettsiae by PCR targeting a partial sequence of the ompA gene followed by sequence analysis. Two additional genes, 17 kDa and 16SrRNA were also targeted for further characterization of the detected Rickettsia species. Rickettsial DNA was detected in 148 out of the 867 (17%) tested ticks. The infection rates in Rh. turanicus, Rh. sanguineus, H. adleri, H. parva, H. dromedarii, and H. impeltatum ticks were 41.7, 11.6, 16.7, 16.2, 11.8 and 20%, respectively. None of the ticks, belonging to the species Rh. bursa and H. aegyptium, were infected. Four SFG rickettsiae were identified: Rickettsia massiliae, Rickettsia africae, Candidatus Rickettsia barbariae and Candidatus Rickettsia goldwasserii.

Significance

The results of this study demonstrate the geographic distribution of SFG rickettsiae and clearly indicate the presence of at least four of them in collected ticks. Palestinian clinicians should be aware of emerging tick-borne diseases in the West Bank, particularly infections due to R. massiliae and R. africae.     

Rickettsia Phylogenomics: Unwinding the Intricacies of Obligate Intracellular Life

Background

Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular α-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs) of open reading frames (ORFs) will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs) from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs).

Methodology/Principal Findings

We present 1823 representative (no gene duplications) and 259 non-representative (at least one gene duplication) rickettsial OGs. While the highly reductive (∼1.2 MB) Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group), TG (typhus group), TRG (transitional group), and SFG (spotted fever group) rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions.

Conclusion/Significance

Collectively, we determined the relative conservation and distribution of 14354 predicted ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets.     

Proteome Analysis and Serological Characterization of Surface-Exposed Proteins of Rickettsia heilongjiangensis

Background

Rickettsia heilongjiangensis, the agent of Far-Eastern spotted fever (FESF), is an obligate intracellular bacterium. The surface-exposed proteins (SEPs) of rickettsiae are involved in rickettsial adherence to and invasion of host cells, intracellular bacterial growth, and/or interaction with immune cells. They are also potential molecular candidates for the development of diagnostic reagents and vaccines against rickettsiosis.

Methods

R. heilongjiangensis SEPs were identified by biotin-streptavidin affinity purification and 2D electrophoreses coupled with ESI-MS/MS. Recombinant SEPs were probed with various sera to analyze their serological characteristics using a protein microarray and an enzyme-linked immune sorbent assay (ELISA).

Results

Twenty-five SEPs were identified, most of which were predicted to reside on the surface of R. heilongjiangensis cells. Bioinformatics analysis suggests that these proteins could be involved in bacterial pathogenesis. Eleven of the 25 SEPs were recognized as major seroreactive antigens by sera from R. heilongjiangensis-infected mice and FESF patients. Among the major seroreactive SEPs, microarray assays and/or ELISAs revealed that GroEL, OmpA-2, OmpB-3, PrsA, RplY, RpsB, SurA and YbgF had modest sensitivity and specificity for recognizing R. heilongjiangensis infection and/or spotted fever.

Conclusions

Many of the SEPs identified herein have potentially important roles in R. heilongjiangensis pathogenicity. Some of them have potential as serodiagnostic antigens or as subunit vaccine antigens against the disease.     

Variations of plasmid content in Rickettsia felis

Background

Since its first detection, characterization of R. felis has been a matter of debate, mostly due to the contamination of an initial R. felis culture by R. typhi. However, the first stable culture of R. felis allowed its precise phenotypic and genotypic characterization, and demonstrated that this species belonged to the spotted fever group rickettsiae. Later, its genome sequence revealed the presence of two forms of the same plasmid, physically confirmed by biological data. In a recent article, Gillespie et al. (PLoS One. 2007;2(3):e266.) used a bioinformatic approach to refute the presence of the second plasmid form, and proposed the creation of a specific phylogenetic group for R. felis.

Methodology/Principal Findings

In the present report, we, and five independent international laboratories confirmed unambiguously by PCR the presence of two plasmid forms in R. felis strain URRWXCal₂ ^T, but observed that the plasmid content of this species, from none to 2 plasmid forms, may depend on the culture passage history of the studied strain. We also demonstrated that R. felis does not cultivate in Vero cells at 37°C but generates plaques at 30°C. Finally, using a phylogenetic study based on 667 concatenated core genes, we demonstrated the position of R. felis within the spotted fever group.

Significance

We demonstrated that R. felis, which unambiguously belongs to the spotted fever group rickettsiae, may contain up to two plasmid forms but this plasmid content is unstable.     

Molecular Evidence for the Presence of Rickettsia Felis in the Feces of Wild-living African Apes

Background

Rickettsia felis is a common emerging pathogen detected in mosquitoes in sub-Saharan Africa. We hypothesized that, as with malaria, great apes may be exposed to the infectious bite of infected mosquitoes and release R. felis DNA in their feces.

Methods

We conducted a study of 17 forest sites in Central Africa, testing 1,028 fecal samples from 313 chimpanzees, 430 gorillas and 285 bonobos. The presence of rickettsial DNA was investigated by specific quantitative real-time PCR. Positive results were confirmed by a second PCR using primers and a probe targeting a specific gene for R. felis. All positive samples were sequenced.

Results

Overall, 113 samples (11%) were positive for the Rickettsia-specific gltA gene, including 25 (22%) that were positive for R. felis. The citrate synthase (gltA) sequence and outer membrane protein A (ompA) sequence analysis indicated 99% identity at the nucleotide level to R. felis. The 88 other samples (78%) were negative using R. felis-specific qPCR and were compatible with R. felis-like organisms.

Conclusion

For the first time, we detected R. felis in wild-living ape feces. This non invasive detection of human pathogens in endangered species opens up new possibilities in the molecular epidemiology and evolutionary analysis of infectious diseases, beside HIV and malaria.     

Western-blot detection of RickA within spotted fever group rickettsiae using a specific monoclonal antibody

Spotted fever group rickettsiae are obligate intracellular pathogens able to manipulate the actin cytoskeleton, thus enabling cell-to-cell spreading during infection. While the RickA protein, which has similarity to the WASP family of Arp2/3-complex activators, was described as being responsible for actin-based motility, recent studies demonstrated that another factor, still unidentified, is also involved in this phenomenon. Here, using recombinant protein of Rickettsia conorii as an antigen, we produced a monoclonal antibody (mAb) directed against RickA. Its specificity was checked using two-dimensional polyacrylamide gel electrophoresis coupled with MS analysis. In Western-blot assays, our antibody recognized the RickA protein from all spotted fever group rickettsiae tested. This mAb would be useful to monitor the expression of RickA in spotted fever group rickettsiae grown under various culture conditions, associated or not with the motility phenotype, and thus to gain better knowledge about the molecular mechanisms involved in their cell-to-cell spreading.     

The lactic acid bacterium Pediococcus acidilactici suppresses autoimmune encephalomyelitis by inducing IL-10-producing regulatory T cells

Background

Certain intestinal microflora are thought to regulate the systemic immune response. Lactic acid bacteria are one of the most studied bacteria in terms of their beneficial effects on health and autoimmune diseases; one of which is Multiple sclerosis (MS) which affects the central nervous system. We investigated whether the lactic acid bacterium Pediococcus acidilactici, which comprises human commensal bacteria, has beneficial effects on experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

Methodology/Principal Findings

P. acidilactici R037 was orally administered to EAE mice to investigate the effects of R037. R037 treatment suppressed clinical EAE severity as prophylaxis and therapy. The antigen-specific production of inflammatory cytokines was inhibited in R037-treated mice. A significant increase in the number of CD4⁺ Interleukin (IL)-10-producing cells was observed in the mesenteric lymph nodes (MLNs) and spleens isolated from R037-treated naive mice, while no increase was observed in the number of these cells in the lamina propria. Because only a slight increase in the CD4⁺Foxp3⁺ cells was observed in MLNs, R037 may primarily induce Foxp3⁻ IL10-producing T regulatory type 1 (Tr1) cells in MLNs, which contribute to the beneficial effect of R037 on EAE.

Conclusions/Significance

An orally administered single strain of P. acidilactici R037 ameliorates EAE by inducing IL10-producing Tr1 cells. Our findings indicate the therapeutic potential of the oral administration of R037 for treating multiple sclerosis.     

Rickettsia monacensis sp. nov., a Spotted Fever Group Rickettsia, from Ticks (Ixodes ricinus) Collected in a European City Park

We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/Munich^T) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.     

Rickettsia Species in African Anopheles Mosquitoes

Background

There is higher rate of R. felis infection among febrile patients than in healthy people in Sub-Saharan Africa, predominantly in the rainy season. Mosquitoes possess a high vectorial capacity and, because of their abundance and aggressiveness, likely play a role in rickettsial epidemiology.

Methodology/Principal Findings

Quantitative and traditional PCR assays specific for Rickettsia genes detected rickettsial DNA in 13 of 848 (1.5%) Anopheles mosquitoes collected from Côte d’Ivoire, Gabon, and Senegal. R. felis was detected in one An. gambiae molecular form S mosquito collected from Kahin, Côte d’Ivoire (1/77, 1.3%). Additionally, a new Rickettsia genotype was detected in five An. gambiae molecular form S mosquitoes collected from Côte d’Ivoire (5/77, 6.5%) and one mosquito from Libreville, Gabon (1/88, 1.1%), as well as six An. melas (6/67, 9%) mosquitoes collected from Port Gentil, Gabon. A sequence analysis of the gltA, ompB, ompA and sca4 genes indicated that this new Rickettsia sp. is closely related to R. felis. No rickettsial DNA was detected from An. funestus, An. arabiensis, or An. gambiae molecular form M mosquitoes. Additionally, a BLAST analysis of the gltA sequence from the new Rickettsia sp. resulted in a 99.71% sequence similarity to a species ({"type":"entrez-nucleotide","attrs":{"text":"JQ674485","term_id":"386870496","term_text":"JQ674485"}}JQ674485) previously detected in a blood sample of a Senegalese patient with a fever from the Bandafassi village, Kedougou region.

Conclusion

R. felis was detected for the first time in An. gambiae molecular form S, which represents the major African malaria vector. The discovery of R. felis, as well as a new Rickettsia species, in mosquitoes raises new issues with respect to African rickettsial epidemiology that need to be investigated, such as bacterial isolation, the degree of the vectorial capacity of mosquitoes, the animal reservoirs, and human pathogenicity.     

Detection of Rickettsia spp in Ticks by MALDI-TOF MS

Background

Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been shown to be an effective tool for the rapid identification of arthropods, including tick vectors of human diseases.

Methodology/Principal Findings

The objective of the present study was to evaluate the use of MALDI-TOF MS to identify tick species, and to determine the presence of rickettsia pathogens in the infected Ticks. Rhipicephalus sanguineus and Dermacentor marginatus Ticks infected or not by R. conorii conorii or R. slovaca, respectively, were used as experimental models. The MS profiles generated from protein extracts prepared from tick legs exhibited mass peaks that distinguished the infected and uninfected Ticks, and successfully discriminated the Rickettsia spp. A blind test was performed using Ticks that were laboratory-reared, collected in the field or removed from patients and infected or not by Rickettsia spp. A query against our in-lab arthropod MS reference database revealed that the species and infection status of all Ticks were correctly identified at the species and infection status levels.

Conclusions/Significance

Taken together, the present work demonstrates the utility of MALDI-TOF MS for a dual identification of tick species and intracellular bacteria. Therefore, MALDI-TOF MS is a relevant tool for the accurate detection of Rickettsia spp in Ticks for both field monitoring and entomological diagnosis. The present work offers new perspectives for the monitoring of other vector borne diseases that present public health concerns.     

Modification of Salmonella Typhimurium motility by the probiotic yeast strain Saccharomyces boulardii

Background

Motility is an important component of Salmonella enterica serovar Typhimurium (ST) pathogenesis allowing the bacteria to move into appropriate niches, across the mucus layer and invade the intestinal epithelium. In vitro, flagellum-associated motility is closely related to the invasive properties of ST. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is widely prescribed for the prophylaxis and treatment of diarrheal diseases caused by bacteria or antibiotics. In case of Salmonella infection, S.b-B has been shown to decrease ST invasion of T84 colon cell line. The present study was designed to investigate the impact of S.b-B on ST motility.

Methodology/Principal Findings

Experiments were performed on human colonic T84 cells infected by the Salmonella strain 1344 alone or in the presence of S.b-B. The motility of Salmonella was recorded by time-lapse video microscopy. Next, a manual tracking was performed to analyze bacteria dynamics (MTrackJ plugin, NIH image J software). This revealed that the speed of bacterial movement was modified in the presence of S.b-B. The median curvilinear velocity (CLV) of Salmonella incubated alone with T84 decreased from 43.3 µm/sec to 31.2 µm/sec in the presence of S.b-B. Measurement of track linearity (TL) showed similar trends: S.b-B decreased by 15% the number of bacteria with linear tract (LT) and increased by 22% the number of bacteria with rotator tract (RT). Correlation between ST motility and invasion was further established by studying a non-motile flagella-deficient ST strain. Indeed this strain that moved with a CLV of 0.5 µm/sec, presented a majority of RT and a significant decrease in invasion properties. Importantly, we show that S.b-B modified the motility of the pathogenic strain SL1344 and significantly decreased invasion of T84 cells by this strain.

Conclusions

This study reveals that S.b-B modifies Salmonella''s motility and trajectory which may account for the modification of Salmonella''s invasion.     

Fluid shear stress regulates the invasive potential of glioma cells via modulation of migratory activity and matrix metalloproteinase expression

Background

Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate.

Methodology/Principal Findings

A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.

Conclusions/Significance

Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.     

Gliding motility of Babesia bovis merozoites visualized by time-lapse video microscopy

Background

Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite.

Methodology/Principal Findings

Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion.

Conclusions/Significance

This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding.     

Thioredoxin 80-activated-monocytes (TAMs) inhibit the replication of intracellular pathogens

Background

Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs).

Principal Findings

In this investigation we present evidence for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L. monocytogenes and B. abortus. Further analysis shows that Trx80 activation prevents the escape of GFP-tagged L. monocytogenes into the cytosol, and induces accumulation of the bacteria within the lysosomes. Inhibition of the lysosomal activity by chloroquine treatment resulted in higher replication of bacteria in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome.

Significance

Our results show that Trx80 potentiates the bactericidal activities of professional phagocytes, and contributes to the first line of defense against intracellular bacteria.     

Capulet and Slingshot share overlapping functions during Drosophila eye morphogenesis

Background

CAP/Capulet (Capt), Slingshot (Ssh) and Cofilin/Twinstar (Tsr) are actin-binding proteins that restrict actin polymerization. Previously, it was shown that low resolution analyses of loss-of-function mutations in capt, ssh and tsr all show ectopic F-actin accumulation in various Drosophila tissues. In contrast, RNAi depletion of capt, tsr and ssh in Drosophila S2 cells all affect actin-based lamella formation differently. Whether loss of these three related genes might cause the same effect in the same tissue remains unclear.

Methods

Loss-of-function mutant clones were generated using the MARCM or EGUF system whereas overexpression clones were generated using the Flip-out system. Immunostaining were then performed in eye imaginal discs with clones. FRAP was performed in cultured eye discs.

Results

Here, we compared their loss-of-function phenotype at single-cell resolution, using a sheet of epithelial cells in the Drosophila eye imaginal disc as a model system. Surprisingly, we found that capt and ssh, but not tsr, mutant cells within and posterior to the morphogenetic furrow (MF) shared similar phenotypes. The capt/ssh mutant cells possessed: (1) hexagonal cell packing with discontinuous adherens junctions; and (2) largely complementary accumulation of excessive phosphorylated myosin light chain (p-MLC) and F-actin rings at the apical cortex. We further showed that the capt/ssh mutant phenotypes depended on the inactivation of protein kinase A (PKA) and activation of Rho.

Conclusions

Although Capt, Ssh and Tsr were reported to negatively regulate actin polymerization, we found that Capt and Ssh, but not Tsr, share overlapping functions during eye morphogenesis.