First-time isolation and quantification of Basidiobolus spp. from reptile faeces in KwaZulu-Natal (South Africa) using selective media.

Members of the genus Basidiobolus are potentially pathogenic fungi, known to cause mycoses in tropical and subtropical countries. Basidiobolus spp. can be associated with animals, and reptiles and amphibians are candidate vectors for the distribution of this fungus. The presence of Basidiobolus spp. was described for different reptiles in several African countries, although not for South Africa. In addition, quantitative data are scarce. The aim of this study was to analyse faeces of selected South African reptiles for the presence and quantity of "viable Basidiobolus units." Faecal samples of gecko and agama lizards were collected and analysed using spread plating, with confirmation by PCR. The addition of dichloran and benomyl to standard fungal media improved the selectivity and allowed quantification of Basidiobolus spp. in reptile faeces. The amount of Basidiobolus spp. varied between 300 and 1.4 × 106  CFU per gram of pooled gecko faeces, which mostly corresponds to >1000 CFU per outside dropping and <100 CFU per inside dropping. About 60% of analysed agama faeces carried Basidiobolus spp., ranging from 150 to 1.2 × 105  CFU per dropping. Our results show for the first time that faeces of South African reptiles frequently carry Basidiobolus spp., confirming that they can contribute to the distribution of this fungus.

A novel role for Celf1 in vegetal RNA localization during Xenopus oogenesis.

The localization of certain mRNAs to the vegetal cortex of Xenopus oocytes is of crucial importance for germ cell development and early embryonic patterning. Vegetal RNA localization is mediated by cis-acting RNA localization elements (LE). Several proteins assemble on the RNA LE and direct transport to the vegetal cortex. Although a number of localization RNP components have been identified, their full composition is unknown. In an RNA affinity purification approach, using the dead end 1 (dnd1) RNA LE, we identified Xenopus Celf1 as a novel component of vegetal localization RNP complexes. Celf1 is part of an RNP complex together with known vegetal localization factors and shows specific interactions with LEs from several but not all vegetally localizing RNAs. Immunostaining experiments reveal co-localization of Celf1 with vegetally localizing RNA and with known localization factors. Inhibition of Celf1 protein binding by localization element mutagenesis as well as Celf1 overexpression interfere with vegetal RNA localization. These results argue for a role of Celf1 in vegetal RNA localization during Xenopus oogenesis.

Draft genome sequences of two ß-glucuronidase positive strains of Salmonella enterica subspecies salamae isolated from reptile feces in KwaZulu-Natal, South Africa.

Two Salmonella enterica isolates obtained from reptile feces displayed ß-glucuronidase activity. Nearly complete genome sequences were obtained after shotgun sequencing and de novo genome assembly. By comparison to reference genomes, both isolates were identified as Salmonella enterica subspecies salamae with the sequence type identified as 1208 and the serotype as 42:r:-.

First draft genome sequence of a Pectobacterium polaris strain isolated in South Africa from potato tuber affected by soft rot.

A phytopathogenic strain of Pectobacterium polaris (designated SRB2) was isolated for the first time in South Africa from a potato tuber affected by soft rot. The draft genome of strain SRB2 encodes various plant cell wall-degrading enzymes and genes associated with biofilm formation and virulence. Antibiotic resistance genes were not detected.

Long-Term Monitoring of the Seasonal Abundance of Basidiobolus spp. in Gecko Feces in KwaZulu-Natal (South Africa).

The fungal genus Basidiobolus is typically associated with ectothermic animals such as amphibians and reptiles. In rare cases, it can cause infections in humans, which are often misdiagnosed. Although usually restricted to tropical and subtropical countries, infections have recently been more frequently reported in hot-dry regions such as Arizona and Saudi Arabia. Reptiles such as geckos are known to shed Basidiobolus spp. via feces and frequently live in close proximity to humans. To establish the frequency and burden of Basidiobolus spp. released by geckos in a suburban location, we regularly quantified viable Basidiobolus units per gram of feces from indoors and outdoors over 3.5 years between 2018 and 2022 using a selective medium. Geckos were shedding Basidiobolus spp. in all seasons, with most counts established ranging between 5.0 and 6.5 log10 cfu per gram. Statistically significant seasonal differences per location were only observed for the outside winter counts and, apparently, correlated to lower temperatures, while inside counts showed no seasonal difference. Overall, counts for droppings collected outdoors were significantly higher than counts for droppings collected indoors. Our data confirm that geckos, which frequently enter homes and are global invaders, are a regular source of this fungus.

Functional dissection of the RNA signal sequence responsible for vegetal localization of XGrip2.1 mRNA in Xenopus oocytes.

Grip2.1 is a conserved PDZ-domain protein with a function in the context of primordial germ cell development and migration in Xenopus embryos. Its mRNA is maternally supplied and found to be associated with the germ plasm, located at the tip of the vegetal cortex in Xenopus oocytes. Here, we demonstrate that the 3'-UTR of XGrip2.1 contains a 211 nucleotide RNA signal sequence that promotes localization to the mitochondrial cloud via the early localization pathway upon injection into stage I oocytes. The same element is also capable of using the late transport pathway if injected into stage III/IV oocytes. In vitro protein interaction studies reveal binding to ElrA/B, Vg1RBP and VgRBP60, proteins that have previously been associated with the vegetal localization machinery. Mutational interference with Vg1RBP and VgRBP60 binding severely reduces early and late localization activity. Selective interference with Vg1RBP binding significantly reduces late localization while having only a mild effect on localization to the mitochondrial cloud, indicating that the signal sequences and protein machinery required for early and late pathway localization though overlapping are not identical.

Identification of vegetal RNA-localization elements in Xenopus oocytes.

Localized mRNAs have been identified in a large variety of cell types where they contribute to the establishment of cell asymmetries and can function as cell fate determinants. In Xenopus, RNAs that localize to the vegetal cortex during oogenesis function in early embryonic patterning as well as in the development of primordial germ cells. Based on their temporal and spatial localization patterns, vegetally localizing RNAs are referred to as either early-pathway RNAs which transiently localize in the mitochondrial cloud, or as late-pathway RNAs. Vegetal RNA-localization is driven by cis-acting signal sequences that, in most cases, were found to reside in the 3'-UTRs and which are recognized by trans-acting localization factors. Here we describe the methods of how vegetal RNA-localization elements can be identified by injection of fluorescently-labeled or tagged RNAs.

Differentiation of Basidiobolus spp. Isolates: RFLP of a Diagnostic PCR Amplicon Matches Sequence-Based Classification and Growth Temperature Preferences.

The genus Basidiobolus, known since 1886, is primarily associated with reptiles and amphibians. Although globally distributed, rare infections caused by members of this genus mainly occur in tropical and subtropical regions. Morphological and physiological characteristics were used in the past for the description of species. However, some of these characteristics vary depending on culture conditions. Therefore, most species names are regarded as synonyms of B. ranarum as the only pathogenic species. Yet, not all environmental isolates are necessarily pathogenic. This study aimed to analyze if environmental Basidiobolus isolates can be distinguished reliably based on morpho-physiological and molecular characteristics. Eleven isolates originally obtained from feces of south African reptiles and one type strain, Basidiobolus microsporus DSM 3120, were examined morpho-physiologically. Sequence analysis of the 18S and partial 28S rRNA gene and restriction analysis of a diagnostic amplicon (restriction fragment length polymorphism, RFLP) were performed for all 12 strains. Based on the results obtained, morphological features and the 18S rRNA sequence proved insufficient for the reliable differentiation of isolates. However, isolates were distinguishable by growth temperature profiles, which matched isolate clusters established by partial 28S rRNA gene sequence and restriction analysis of a Basidiobolus specific diagnostic PCR amplicon. Our results indicate that RFLP analysis can be used as a fast screening method to identify Basidiobolus isolates with similar physiological characteristics.

Loss of inner hair cell ribbon synapses and auditory nerve fiber regression in Cldn14 knockout mice.

Proper functioning of the auditory nerve is of critical importance for auditory rehabilitation by cochlear implants. Here we used the Cldn14-/- mouse to study in detail the effects of Claudin 14 loss on auditory synapses and the auditory nerve. Mutations in the tight junction protein Claudin 14 cause autosomal recessive non-syndromic hearing loss (DFNB29) in humans and mice, due to extensive degeneration of outer and inner hair cells. Here we show that massive inner hair cell loss in Cldn14-/- mice starts after the third postnatal week. Immunohistochemical analysis, using presynaptic Ribeye and postsynaptic GluR2 or PSD 95 as markers, revealed the degeneration of full ribbon synapses in inner hair cells from apical cochlear regions already at postnatal day 12 (P12). At P20, significant reduction in number of ribbon synapses has been observed for all cochlear regions and the loss of synaptic ribbons becomes even more prominent in residual inner hair cells from middle and apical cochlear regions at P45, which by then lost more than 40% of all ribbon synapses. In contrast to excessive noise exposure, loss of Claudin 14 does not cause an increase in "orphan" ribbons with no postsynaptic counterpart due to a reduction of postsynaptic structures. Hair cell loss in Cldn14-/- mice is associated with regression of peripheral auditory nerve processes, especially of outer radial fibers, which normally innervate the outer hair cells. The number of spiral ganglion neurons per area, however, was unchanged between the genotypes. Different effects were observed in the cochlear nucleus complex (CNC), the central projection area of the auditory nerve. While the dorsal cochlear nucleus (DCN) showed a significant 19.7% volume reduction, VGLUT-1 input was reduced by 34.4% in the ventral cochlear nucleus (VCN) but not in the DCN of Cldn14-/- mice. Taken together, massive inner hair cell loss starts after the third postnatal week in Cldn14-/- mice, but is preceded by the loss of ribbon synapses, which may be a first sign of an ongoing degeneration process in otherwise morphologically inconspicuously inner hair cells. In addition to the regression of peripheral nerve processes, reduced levels of VGLUT-1 in the VCN of Cldn14-/- mice suggests that Claudin 14 loss does not only cause hair cell loss but also affects peripheral and central connectivity of the auditory nerve.

Functional characterization of Drosophila Translin and Trax.

The vertebrate RNA and ssDNA-binding protein Translin has been suggested to function in a variety of cellular processes, including DNA damage response, RNA transport, and translational control. The Translin-associated factor X (Trax) interacts with Translin, and Trax protein stability depends on the presence of Translin. To determine the function of the Drosophila Translin and Trax, we generated a translin null mutant and isolated a trax nonsense mutation. translin and trax single and double mutants are viable, fertile, and phenotypically normal. Meiotic recombination rates and chromosome segregation are also not affected in translin and trax mutants. In addition, we found no evidence for an increased sensitivity for DNA double-strand damage in embryos and developing larvae. Together with the lack of evidence for their involvement in DNA double-strand break checkpoints, this argues against a critical role for Translin and Trax in sensing or repairing such DNA damage. However, Drosophila translin is essential for stabilizing the Translin interaction partner Trax, a function that is surprisingly conserved throughout evolution. Conversely, trax is not essential for Translin stability as trax mutants exhibit normal levels of Translin protein.