Single-cell RNA-Seq reveals intracellular microbial diversity within immune cells during SARS-CoV-2 infection and recovery, 2023, Sunita Yadav et al

[Mij]

Highlights

• 76 bacterial species of 15 genera across healthy, COVID-19-positive, and recovered individuals
• Higher abundance of opportunistic intracellular bacteria in recovered group
• Higher microbial abundance within antigen-presenting cells of COVID-19 patients
• Differential microbial abundance in Treg, memory T, and platelets in COVID-19

Summary
Intracellular microorganisms, like viruses, bacteria, and fungi, pose challenges in detection due to their non-culturable forms. Transcriptomic analysis at cellular level enables exploration of distributions and the impact of these microorganisms on host cells, a domain that remains underexplored because of methodological limitations.

Single-cell technology shows promise in addressing this by capturing polyadenine-tailed transcripts, because recent studies confirmed polyadenylation in microbial transcriptomes. We utilized single-cell RNA-seq from PBMCs to probe intracellular microbes in healthy, SARS-CoV-2-positive, and recovered individuals.

Among 76 bacterial species detected, 16 showed significant abundance differences. Buchnera aphidicola, Streptomyces clavuligerus, and Ehrlichia canis emerged significantly in memory-B, Naïve-T, and Treg cells. Staphylococcus aureus, Mycoplasma mycoides, Leptospira interrogans, and others displayed elevated levels in SARS-CoV-2-positive patients, suggesting possible disease association.

This highlights the strength of single-cell technology in revealing potential microorganism’s cell-specific functions. Further research is essential for functional understanding of their cell-specific abundance across physiological states.

https://www.cell.com/iscience/fullt...m/retrieve/pii/S2589004223024343?showall=true

 

[SNT Gatchaman]

Introduction

The presence of microbes, either intracellular or extracellular, significantly impacts how they interact with the host’s immune system and their capacity to evade immune defenses. [...] intracellular microbes may inhabit specific host cell compartments such as the endosome or cytosol, where they secure vital nutrients and evade direct antibody attacks. In some cases, neutrophils, fibroblasts, or epithelial cells can also serve as habitat for intracellular pathogens.

when it comes to studying the presence of intracellular microbes, the conventional techniques tend to provide an average genome output that can obscure crucial insights. In contrast, single-cell sequencing offers a unique and powerful window into the complex world of individual cells, particularly in the context of studying microbial presence in the blood PBMCs.

This investigation centered on microbial communities within PBMCs from three groups: healthy individuals, SARS-CoV-2-positive individuals, and those who have recovered. The aim was to identify and elucidate the diversity of intracellular microbes found within the definitive cell types using scRNA-Seq technology and to uncover potential associations between microbial presence and host cellular responses at different physiological conditions.

Toward that, we identified 76 microbes enriched within 12 distinct immune cell types. Of these, 16 microbes were differentially abundant between different across the healthy, positive, and recovered groups. A closer look at the cell-type wise abundance of these microbes revealed 8 differentially expressed bacterial species across the groups in 6 different cell types.

 

[SNT Gatchaman]

Differential presence of non-human reads in healthy, SARS-CoV-2-positive, and recovered individuals

We selected 27 individuals (14 COVID-19-positive, 10 recovered, and 3 healthy)

We identified 12 different cell clusters, viz. naive T cell, naive B cell, classical monocytes, neutrophil, natural killer (NK) cell, memory B cell, macrophages, memory T cell, Treg cell, dendritic cells (DC), platelets, and plasma cells across the three groups. A total of ~11 billion reads were generated from 97,103 cells, of which 9 billion reads mapped to the human genome, whereas nearly 2 billion reads remained unmapped.

The differential proportions of human and non-human reads found in each group were as follows: healthy (82.98% human versus 17.02% non-human), positive (82.21% human versus 17.79% non-human), and recovered (77.96% human versus 22.09% non-human), wherein we identified a significant differential distribution of non-human reads per 1000 human reads across the three groups (p value < 0.001).

Overall, we identified a total of 76 bacterial species across the 15 genera and 3 groups, which were further analyzed at single-cell resolution for their cell-type-specific abundance and species-specific functions across the three groups.

 

[SNT Gatchaman]

Differential abundance of bacterial species in COVID-19-infected and recovered individuals versus healthy individuals

Because all the 76 microbes were detected in the 3 groups, no significant difference in Shannon alpha diversity was observed. However, our beta diversity analysis [...] unveiled distinctive cluster patterns, indicating that despite having the same number of microbial species, they are differentially abundant between the three groups. Subsequent adonis tests were performed to validate pronounced dissimilarities of microbial abundance across the three groups (p value = 0.003).

An important finding from our analysis was that all the three bacterial species that are highly abundant in the recovered (B. aphidicola, C. beijerinckii, and E. canis) are opportunistic in nature. The increased abundance of opportunistic bacteria in the recovered might be attributed to the dysfunctional T cell response, as T cell dysfunction is associated with suppressed innate immune response.

Intriguingly, C. beijerinckii and E. canis are reported to be associated with inflammatory response, which might be one of the contributory factors for the increased inflammatory response, post-recovery. The findings highlight the intricate and complex nature of host microbe interplay during infection and recovery and importance of understanding the cell-type-specific microbial diversity.

 

[SNT Gatchaman]

Immune-cell-type-specific significant bacteria and their potential roles

all these 6 cell types are involved in the antigen presentation process. Our analysis revealed that although naive T and Treg cells have similar relative abundance across the three groups, microbial abundance within these cells across the three groups are strikingly different. Although higher microbial abundance in the antigen-presenting cells is in line with the function of these cells, differential microbial abundance in cell types such as platelets, memory T, and Treg needs closer attention for functional relevance.

Additionally, we observed microbial abundance in the classical monocyte, NK cell, and plasma cells exclusively in the SARS-CoV-2-positive patients. Although classical monocytes and NK cells are known to be involved in the antigen presentation process, the role of plasma cells during COVID-19 needs context-specific exploration/elucidation.

Of particular interest is E. canis, an intracellular bacterium, which was found to be significantly enriched in the naive T cells and Treg of the positive patients compared with the healthy individuals. Additionally, it was observed to be more abundant in neutrophils of the recovered compared with the positive. This suggests a plausible unique role of E. canis in the T-cell-mediated immune response of COVID-19-positive patients. Since it is known to cause apoptosis in monocytes, the role of this bacteria in T cell biology and function could be explored in the future.

The presence of specific microorganisms within specific cell types plausibly suggests an interaction between the host and the differential abundance of the microbes. Going forward, further research is required to grasp the underlying biological mechanisms at play.

 

[SNT Gatchaman]

Discussion

The intricate interplay between the disease severity and presence of microbes within the immune cell types, shedding light on host immune responses during infectious diseases, remains a largely uncharted domain. [...] This study pioneers the investigation of bacterial profiles within the immune cells at a single-cell resolution across different health states and augments important insights.

Although the immune system guards against microbial intrusion to curtail infections, some microbes manage to infiltrate and persist within the body. Consequently, dysbiosis at different taxa levels and their niches in the host body can affect immunity and disease outcomes, underscoring the importance of investigating microbial presence and their impact on immune cell dysregulation.

Notably, the positive and recovered groups exhibited an enrichment of potential pathogenic species, whereas the healthy individuals showcased a prevalence of specific commensal counterparts. Particularly intriguing were the findings related to S. clavuligerus and S. pristinaespiralis—both commensal inhabitants within the healthy individuals. These species, known for producing beneficial antibiotics, clavulanic acid and pristinamycin, could signify a role in strengthening immune defenses. In contrast, a decline in these commensal species was noted in the recovered and positive patients.

Remarkably, S. clavuligerus and S. pristinaespiralis exhibited higher prevalencein memory B and naive B cells of healthy individuals, suggesting a potential reservoir role. Memory B cells are known for their long-lasting immune memory and long life. This contribution could foster harmonious microbiota persistence within a robust immune system. In a parallel observation, E. canis was more prevalent in both Tregs and naive T cells, whereas E. albertii displayed greater detection in the naive B cells among SARS-CoV-2-positive patients compared with the recovered individuals. This presence within the immune cells suggests pathogen evasion, given these cells’ role in infection defense and immune response activation.

Interestingly, P. tolaasii was enriched in the platelet cells of healthy individuals compared with the positive patients, displaying a counterintuitive trend compared with the group-level pattern.

To validate our findings at the group level, we investigated an additional dataset of COVID-19 patients from a separate geographical region and discovered the presence of 8 bacterial species in the PBMCs, coinciding with our results. [...] Notably, within this overlap, 3 potential pathogenic species demonstrated significant enrichment in the positive patients within our cohort. This leads to a noteworthy observation that microbes can be found in PBMCs not only during infection but also in healthy and recovered individuals.

despite finding these intriguing microbial footprints within the immune cells, a significant question remains shrouded in uncertainty about the precise role played by these microbes within these immune cells, which awaits further rigorous investigation.

A follow-up study investigating the same not only in PBMCs but also in other tissue types is warranted. Furthermore, although our study focused on COVID-19, we encourage similar research to be done in the context of other infectious diseases.

 

[SNT Gatchaman]

This study needs to be replicated in healthy vs recovered/asymptomatic vs LC ± vs acute.