Van Mellaert, L. | Search bioRxiv

bioRxiv2023

Enhanced protein secretion in reduced genome strains of Streptomyces lividans

S. lividans TK24 is a popular host for the production of small molecules and for the secretion of heterologous proteins. TK24 has a large genome with at least 29 secondary metabolite gene clusters that are non-essential for viability and undergo complex regulation. To optimize heterologous protein secretion, we previously constructed ten chassis strains that are devoid of several secondary metabolite gene clusters. Genome reduction was aimed at reducing carbon flow to secondary metabolites and pigmentation in the spent growth medium and improving colony morphology. Strains RG1.0-RG1.10 contain various deletion combinations of the blue actinorhodin cluster (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red) and coelimycin A (cpk) clusters, the melanin cluster (mel), the matAB genes that affect mycelial aggregation and the non-essential sigma factor hrdD that controls the transcription of Act and Red regulatory proteins. Two derivative strains, RG1.5 and 1.9, showed a [~]15% reduction in growth rate, >2-fold increase in the total mass yield of their native secretome and altered abundance of several specific proteins compared with TK24. Metabolomics and RNAseq analysis revealed that genome reduction led to rapid cessation of growth due to aminoacid depletion and caused both redox and cell envelope stresses, upregulation of the Sec-pathway components secDF and chaperones and a cell envelope two component regulator. RG1.9 maintained elevated heterologous secretion of mRFP and mTNF by 12-70%. An integrated model is presented linking genome reduction and enhanced secretion.

systems biology↗

bioRxiv2021

Alphavirus and flavivirus infectivity is disrupted by components of the bacterial cell wall

The impact of the host microbiota on arbovirus infections is currently not well understood. Arboviruses are viruses transmitted through the bites of infected arthropods, predominantly mosquitoes or ticks. The first site of arbovirus inoculation is the biting site in the host skin, which is colonized by a complex microbial community that could possibly influence arbovirus infection. We demonstrated that pre-incubation of arboviruses with certain components of the bacterial cell wall, including lipopolysaccharides (LPS) of some Gram-negative bacteria and lipoteichoic acids or peptidoglycan of certain Gram-positive bacteria, significantly reduced arbovirus infectivity in vitro. This inhibitory effect was observed for arboviruses of different virus families, including chikungunya virus of the Alphavirus genus and Zika virus of the Flavivirus genus, showing that this is a broad phenomenon. A modest inhibitory effect was observed following incubation with a panel of heat-inactivated bacteria, including bacteria residing on the skin. No viral inhibition was observed after pre-incubation of cells with LPS. Furthermore, a virucidal effect of LPS on viral particles was noticed by electron microscopy. Therefore, the main inhibitory mechanism seems to be due to a direct effect on the virus particles. Together, these results suggest that bacteria are able to decrease the infectivity of alphaviruses and flaviviruses. ImportanceDuring the past decades the world has experienced a vast increase in epidemics of alphavirus and flavivirus infections. These viruses can cause severe diseases such as hemorrhagic fever, encephalitis and arthritis. Several alpha- and flaviviruses, such as chikungunya virus, Zika virus and dengue virus, are significant global health threats because of their high disease burden, their widespread (re-)emergence and the lack of (good) anti-arboviral strategies. Despite the clear health burden, alphavirus and flavivirus infection and disease are not fully understood. A knowledge gap in the interplay between the host and the arbovirus is the potential interaction with host skin bacteria. Therefore, we studied the effect of (skin) bacteria and bacterial cell wall components on alphavirus and flavivirus infectivity in cell culture. Our results show that certain bacterial cell wall components markedly reduced viral infectivity by directly interacting with the virus particle.

microbiology↗