# Identification of Sialidase-Producing Bacteria in the Tsetse Gut and Characterisation of a Paenibacillus Sialidase: A Potential Tool for Paratransgenic Control of African Trypanosomes

## TL;DR

This study identifies bacteria in tsetse flies that produce sialidase enzymes, which could help control African trypanosomes by interfering with parasite survival.

## Contribution

The discovery of a Paenibacillus sialidase with dual pH optima and potential for paratransgenic control of trypanosomes is novel.

## Key findings

- Sialidase activity was detected only in tsetse samples from Cameroon.
- A Paenibacillus sialidase gene was identified, cloned, and characterized with specific enzymatic properties.
- The sialidase enzyme shows potential as a tool for paratransgenic control of trypanosome colonization.

## Abstract

The gut microbiota of Tsetse influences several aspects of the host physiology and vector competence. Trypanosome survival in the tsetse midgut depends on surface sialylation. We hypothesised that bacterial sialidases in the tsetse gut could interfere with parasite transmission by counteracting trypanosomal trans-sialidase activity. This study aimed to detect sialidase activity within the tsetse gut, isolate sialidase-producing bacterial strains, and to characterise the enzymatic properties of bacterial sialidases. Tsetse collected from five African countries (Cameroon, Chad, Ethiopia, Ghana and Nigeria) were screened for sialidase activity using a newly developed field-compatible assay. Detectable activity was found only in samples from Cameroon. Bacterial isolates from these samples, representing several genera (Bacillus, Stenotrophomonas, Pseudomonas, Paenibacillus, Enterobacter), were screened for sialidase production. Whole-genome sequencing identified a sialidase gene in a Paenibacillus species. The gene was cloned, expressed in E. coli, and the recombinant enzyme was purified and biochemically characterised. The sialidase exhibited a molecular weight of approximately 120 kDa, showed dual pH optima at pH 5 and 7. Enzymatic activity was enhanced by Cu2+ ions, and kinetic analysis revealed a vmax of 2.514 nmol/mg/min and a KM of 332.7 μM for the synthetic substrate 4-methylumbelliferyl-N-acetylneuraminic acid. These findings indicate that sialidase-producing Paenibacillus and its enzyme may offer promising tools for paratransgenic approaches aimed at limiting trypanosome colonisation in tsetse flies.