In this article, the authors from India and Rwanda used single-cell RNA sequencing (RNA-seq) to investigate intracellular microbes in peripheral blood mononuclear cells (PBMCs) of healthy individuals, in people with SARS-CoV-2, and individuals who recovered from SARS-CoV-2 infection. The aim of the study was to identify the diversity of intracellular microbes in different cell types, and to reveal possible relationships between microbial presence and host cellular responses under different pathophysiological conditions. The results demonstrated diversity of intracellular microbes in immune cells of healthy individuals, SARS-CoV-2 positive individuals, and COVID-19 recovered individuals. Out of the 76 bacterial species tested, the abundance of 16 bacteria species was significantly different in at least one group.
The presence of microbes, whether intracellular or extracellular, has a significant impact on their interaction with the host’s immune system and their ability to evade immune defenses. https://discovermednews.com/the-presence-of-concurrent-intracellular-pathogens-can-lead-to-t-cell-exhaustion-and-potentially-severe-consequences/
It is intriguing that many intracellular pathogens commonly reside within highly efficient antimicrobial defense cells, such as macrophages and dendritic cells, to find shelter from humoral defenses. Bacterial species Mycobacterium Tuberculosis, Staphylococcus aureus and Salmonela can reside within macrophages and perforate the phagosome of macrophages, hijacking the phagosomal processes. (Hiyoshi H, et al. Virulence factors perforate the pathogen-containing vacuole to signal efferocytosis. Cell Host & Microbe 2022; 30, 163–170) https://doi.org/10.1016/j.chom.2021.12.001
Neutrophils, fibroblasts or epithelial cells can also serve as habitat for intracellular pathogens. The intracellular microbes may also inhabit specific host cell compartments, such as the endosome or cytosol, where they evade direct antibody attacks. Understanding the functional characteristics of intracellular pathogens at the cellular level is important to effectively address the obstacles associated with their treatment.
The detection of intracellular microbes presents challenges due to methodological limitations. The authors noted that single-cell sequencing offers a powerful window into the complex world of individual cells, especially in the context of studying microbial presence in the blood PBMCs.
About the study
The authors used single-cell RNA sequencing (RNA-seq) to investigate microbial communities in PBMCs from three groups: healthy people, SARS-CoV-2 positive individuals, and individuals who have recovered from SARS-CoV-2 infection. The study included 27 subjects, 14 of whom were positive for SARS-CoV-2, 10 recovered from COVID-19, and three were healthy.
A total of 97103 cells were captured post filtration, 19026 from healthy individuals, 59792 from SARS-CoV-2 positive individuals, and 18285 from COVID-19 recovered individuals. Approximately 11 billion reads were generated from 97103 cells, of which approximately 9 billion reads were mapped to the human genome, while nearly 2 billion reads were unmapped.
Each group had a differential proportions of human and non-human reads. In healthy people, the proportion was 82.98% human vs. 17.02% non-human. In the group of SARS-CoV-2 positive individuals, the proportion was 82.21% human vs. 17.79% non-human, and in individuals who have recovered from COVID-19 the proportion was 77.96% human vs. 22.09%. The analysis showed that these non-human reads belonged to microbial entities.
The ilustration from original article of Yadav S et al. ISCIENCE 2023
Bacteria were predominantly identified in all groups, accounting for 9.44%, 12.06%, and 15.49% of the reads in the healthy, SARS-CoV-2 positive, and COVID-19 recovered groups, respectively. Archaea were identified in proportions of 6.46%, 4.54%, and 5.39% of the reads in the respective groups. Viruses were detected in 1.12%, 1.19%, and 1.16% of the reads in the respective groups.
A total of 76 bacterial species were further analyzed for their abundance across the three groups. Out of the 76 bacterial species tested, the abundance of 16 bacteria species was significantly different in at least one group (healthy vs. SARS-CoV-2 positive, SARS-CoV-2 positive vs. COVID-19 recovered, healthy vs. COVID-19 recovered).
The results showed higher abundance of Streptomyces pristinaespiralis in healthy individuals (compared to the SARS-CoV-2 positive and COVID-19 recovered), S. clavuligerus (compared to COVID-19 recovered), and Pseudomonas tolaasii (compared to SARS-CoV-2 positive).
The group of individuals who have recovered from SARS-CoV-2 infection had a higher abundance of Buchnera aphidicola, Ehrlichia canis and Clostridium beijerinckii (compared to SARS-CoV-2 positive or healthy individuals). It is important to note that all three bacterial species that were highly abundant in individuals who recovered from COVID-19 are opportunistic (B. aphidicola, C. beijerinckii and E. canis). The authors noted that increased abundance of opportunistic bacteria in individuals who have recovered from COVID-19 may be due to the dysfunctional T cell response. In addition, C. beijerinckii and E. canis are associated with an inflammatory response, and this could be one of the factors contributing to the enhanced inflammatory response after the SARS-CoV-2 infection.
SARS-CoV-2 positive individuals had a higher abundance of three species: Clostridium botulinum, Bacillus thuringiensis, and Staphylococcus aureus than healthy individuals and COVID-19 recovered individuals. According to the authors, these three species are important in the context of disease progression.
The abundance of 16 bacterial species that differed significantly in at least one group was analyzed in 12 different cell types, including naïve T cells, naïve B cells, classical monocytes, neutrophils, natural killer (NK) cells, memory B cells, macrophages, memory T cells, regulatory T cells (Tregs) , dendritic cells (DC), platelets, and plasma cells.
Intracellular microbes were found in 8 cell types of healthy individuals, in 12 cell types of SARS-CoV-2 positive individuals, and in 6 cell types of COVID-19 recovered individuals. In all groups, microbial reads were found in six cell types involved in the antigen presentation process: Tregs, neutrophils, naïve T cells, naïve B cells, macrophages, and DC. This suggests their consistent presence in various health conditions. The finding of different microbial abundance in cell types such as platelets remains to be investigated.
Further analysis revealed that eight bacterial species, namely E. canis, B. aphidicola, S. clavuligerus, S. pristinaespiralis, P. tolaasii, and E. albertii, were expressed in six different cell types in all groups. E. canis was more abundant in the naïve T cells and Tregs of SARS-CoV-2 positive individuals than in those of healthy individuals, and in neutrophils of COVID-19 recovered individuals than in SARS-CoV-2 positive individuals. According to the authors, E. canis has a plausible role in the immune response mediated by T cells.
Bacterial species Buchnera aphidicola, Streptomyces clavuligerus, S. pristinaespiralis, and Pseudomonas tolassi were more abundant in memory B cells, naïve B cells, neutrophils, and platelets of healthy individuals than in those of SARS-CoV-2 positive individuals.
In conclusion, this study demonstrated diversity of intracellular microbes in immune cells of healthy individuals, SARS-CoV-2 positive individuals, and COVID-19 recovered individuals. The results also confirm the importance of single-cell technology in revealing the diversity of intracellular microbes in different cell types. In addition, the identification of specific bacterial species that are associated with different cell types sheds light on the intricate and complex nature of the interplay between the immune system and microbes during infection and recovery.
This article was published in ISCIENCE.
Journal Reference
Yadav, S. et al. Single-cell RNA-Seq reveals intracellular microbial diversity within Immune cells during SARS-CoV-2 Infection and Recovery, ISCIENCE October 27, 2023. (Open Access)Â https://doi.org/10.1016/j.isci.2023.108357