Coronavirus disease 2019 (COVID-19), caused by a novel beta-coronavirus (severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)), has been a global pandemic and caused more than five million deaths worldwide until November of 2021 [1
]. The primary symptoms of COVID-19 are demonstrated in the respiratory system, and extrapulmonary manifestations including gastrointestinal (GI) symptoms, thrombotic complications, and myocardial dysfunction are common [2
]. SARS-CoV-2 could invade human cells via the angiotensin-converting enzyme 2 (ACE2) receptor, which is highly expressed in intestines and plays an important role in maintaining gut health [3
]. The infection of SARS-CoV-2 could impair the normal expression of ACE2, which might result in several adverse outcomes, including GI symptoms as well as the dysbiosis of gut microbiota [6
]. Reports from multiple regions of the world showed that 15% to 69% of COVID-19 patients had at least one GI symptom [7
The microbial communities that reside in the human gut could maintain host homeostasis by providing essential functions, including immunomodulation, nutrient metabolism, and structural protection against pathogenic microorganisms [11
]. Altered gut microbiota was observed among patients with a wide range of infectious diseases, including influenza and other respiratory viral infections [14
]. Recent studies also described the alterations in the gut microbial composition of COVID-19 patients, characterized by enrichment of opportunistic pathogens and depletion of beneficial commensals [18
]. In addition, three bacterial members from the Firmicutes
phylum were positively and two beneficial species, Alistipes onderdonkii
and Faecalibacterium prausnitzii
were inversely associated with COVID-19 severity [21
]. However, the potential mechanism underlying the associations between the gut microbiome and COVID-19 severity remains to be explored.
SARS-CoV-2 infection induces the host immune responses to eliminate the virus, and previous evidence suggested that aberrant immune responses were responsible for adverse outcomes and possibly other inflammations beyond COVID-19 [22
]. The GI tract is the largest immunological organ in the human body and its resident microbiota are known to modulate host immune responses [24
]. According to a prospective study, the gut microbial composition was correlated with the increase of inflammation markers, including interleukin (IL)-10, tumor necrosis factor-α, and C-reactive protein (CRP) in COVID-19 patients [21
]. Nevertheless, data revealing the global relations between the gut microbiome and host systemic immune response in COVID-19 are still limited.
The microbiota-host immune interactions could be mediated by other host factors such as gut barrier function. Intestinal epithelial cells provide a physical and biochemical barrier that segregates host tissue and bacteria to maintain intestinal homeostasis [26
]. Both virus infection and altered gut microbiota could disturb the normal function of the gut barrier and lead to a leaky gut with enhanced gut permeability [27
], which aggravates over-activation of the host immune response [28
]. Thus far, no study has characterized the role of gut barrier dysfunction in the relationship between gut microbes and host immune homeostasis in COVID-19 patients, which may deepen our understanding of COVID-19 pathophysiology.
To better understand the role of gut microbiota in COVID-19 pathogenesis, we characterized the gut microbiota and gut barrier function among 63 COVID-19 patients and 8 uninfected controls through metagenomic and metaproteomic approaches and estimated the associations of gut microbiota with disease severity as well as host systemic immune responses.
It has been known that the gut system is actively involved in COVID-19 pathophysiology for the high expression of ACE2, which is the receptor of SARS-CoV-2 [3
]. The intestinal infection of SARS-CoV-2 could lead to the disruption of the intestinal homeostasis and the host immune homeostasis, which were responsible for the adverse outcomes of COVID-19. In the current study, we observed a significant change in the composition of gut microbiota of COVID-19 patients compared with controls and identified several microbial features at both taxonomic and functional levels associated with COVID-19 severity and host immune responses. Besides, through an integrative analysis of multi-omics data, we found that gut barrier dysfunction might play a role in the crosstalk between gut microbes and host immune homeostasis in COVID-19 patients.
Our results echo the findings from previous studies, reporting the microbial species alterations that were associated with COVID-19 status [19
]. In our study, several commensal species, such as Ba. uniformis
, F. prausnitzii
, and Bi. pseudocatenulatum
, as well as fermentative species, including E. eligens
and Ba. eggerthii
, were depleted in the gut microbiota of COVID-19 patients. Of which, F. prausnitzii
were also found depleted in COVID-19 patients in another Chinese study [21
]. These commensal species could maintain the physical separation between the microorganism and the host and prevent the invasion of pathogens through multiple approaches, including secreting anti-microbial peptides and SCFAs [47
The decrease of commensal species might disturb the normal function of the gut barrier and lead to a leaky gut with enhanced gut permeability [27
]. In the current study, we observed the gut barrier dysfunction characterized by metaproteomic alterations in COVID-19 patients. The increased intestinal permeability was further supported by higher circulating levels of LBP and the detection of bacterial proteins in blood samples in our patients. Lipopolysaccharide is the major outer membrane pathogen-associated molecular pattern of Gram-negative bacteria which can cause an acute inflammatory response by triggering the release of a vast number of inflammatory cytokines [49
]. The leaky gut might promote the transportation of microbes or endotoxins like lipopolysaccharide from the intestine into the blood, which could lead to the immune homeostasis disturbance of COVID-19 patients. Together, these lines of evidence suggested that the alterations of the gut microbiome were associated with SARS-CoV-2 infection and such associations might be mediated by the gut barrier dysfunction in COVID-19 patients.
The dysbiosis of the gut microbiome and dysfunction of the gut barrier could influence the balance between gut microbiota and host, resulting in a worsened inflammation-induced injury [25
]. Over-reaction of the human immune response was the major reason for the poor prognosis of COVID-19 [22
]. In our study, the COVID-19 patients had a more pro-inflammatory gut microbiota profile with several opportunistic pathogens being enriched in patients with mild or severe disease, such as Ba. ovatus
, Ac. bereziniae
, C. innocuum
, Bu. contaminans
, and Ba. Nordii
. In previous reports, Ba. nordii
was found to be associated with COVID-19 [18
]. Multiple virulence genes related to these species were also observed to be more abundant in severe COVID-19 patients. These microbial pathogenic factors could translocate through the leaky gut into the circulating system, promote the secretion of inflammatory cytokines by activating pattern recognition receptor-like TLRs and NOD-like receptors, and therefore lead to systemic inflammation [25
]. In addition, we observed that the abundances of several microbial species changed along with the COVID-19 progression and were associated with biomarkers of host immune and inflammation. For example, Bu. contaminans
were negatively associated with T cell-related transcription modules, which represented T cell activity and were found to reflect the dynamic immune response in COVID-19 [31
]. Bu. contaminans
was also reported in severe respiratory infection [51
]. This pathogenic species could employ a type VI effector to activate the pyrin inflammasome and trigger inflammation [52
], produce tyrosine kinase BceF and phosphotyrosine phosphatase BceD to strengthen the epithelial disruption, and further exacerbate the inflammation [53
]. These results collectively supported a potential role of the gut microbiota in the host immune responses during COVID-19 progression.
Another potential reason for the excessive inflammation in COVID-19 patients might be the enrichment of the glycolysis pathway, which was reported to be associated with higher SRAS-Cov-2 activity [54
]. Under viral and bacterial infections, especially during macrophage polarization and dendritic cell activation, the major energy metabolism switches from lipid towards glycolysis to generate ATP because of the engagement of TLR with the related activation of the PI3K/Akt pathway [55
]. However, the causal relationship between SARS-CoV-2 infection and the enrichment of the glycolysis pathway remains unclear, and further experimental studies are warranted.
To our knowledge, this is the first study to explore the gut microbial-host immunity crosstalk through the integration of metagenomic, proteomic, and metaproteomic approaches. These results, however, should be interpreted with caution. First, because of the case-control study design, we were not able to assess the temporal relationship of the gut microbiota with COVID-19 development. Second, residual confounding from dietary components, physical activity, BMI, and related comorbidities is possible. Third, the number of our included patients and especially the uninfected controls was relatively small, and the sample size for metaproteomic measurement was even smaller. The limited sample size influenced the statistical power of our analysis. Hence, it should be cautious when generalizing our results to other populations. Further large-scale population-based studies are warranted to validate our findings, and intervention studies could help to explore the causal roles of gut microbiota in the pathogenesis underlying COVID-19 development.
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