Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

Universidade Católica Portuguesa

Universidade de Lisboa

 is able to establish a chronic colonization of lung macrophages in a controlled replication manner, giving rise to a so-called latent infection. Conversely, when intracellular bacteria undergo actively uncontrolled replication rates, they provide the switch for the active infection called tuberculosis to occur. Our group found that the pathogen is able to manipulate the activity of endolysosomal enzymes, cathepsins, directly at the level of gene expression or indirectly by regulating their natural inhibitors, cystatins. To provide evidence for the crucial role of cathepsin manipulation for the success of tuberculosis bacilli in their intracellular survival, we used liposomal delivery of saquinavir. This protease inhibitor was previously found to be able to increase cathepsin proteolytic activity, overcoming the pathogen induced blockade. In this study, we demonstrate that incorporation in liposomes was able to increase the efficiency of saquinavir internalization in macrophages, reducing cytotoxicity at higher concentrations. Consequently, our results show a significant impact on the intracellular killing not only to reference and clinical strains susceptible to current antibiotic therapy but also to multidrug- and extensively drug-resistant (XDR) Mtb strains. Altogether, this indicates the manipulation of cathepsins as a fine-tuning strategy used by the pathogen to survive and replicate in host cells.

Liposomal Delivery of Saquinavir to Macrophages Overcomes Cathepsin Blockade by  and Helps Control the Phagosomal Replicative Niches.

The lack of effective therapeutics against emerging multi-drug resistant strains of  () prompts the identification of novel anti-tuberculosis targets. The essential nature of the peptidoglycan (PG) layer of the mycobacterial cell wall, which features several distinctive modifications, such as the -glycolylation of muramic acid and the amidation of D--glutamate, makes it a target of particular interest. To understand their role in susceptibility to beta-lactams and in the modulation of host-pathogen interactions, the genes encoding the enzymes responsible for these PG modifications ( and /, respectively) were silenced in the model organism  using CRISPR interference (CRISPRi). Although beta-lactams are not included in TB-therapy, their combination with beta-lactamase inhibitors is a prospective strategy to treat MDR-TB. To uncover synergistic effects between the action of beta-lactams and the depletion of these PG modifications, knockdown mutants were also constructed in strains lacking the major beta-lactamase of  BlaS, PM965 ( Δ) and PM979 ( Δ Δ). The phenotyping assays affirmed the essentiality of the amidation of D--glutamate to the survival of mycobacteria, as opposed to the -glycolylation of muramic acid. The qRT-PCR assays confirmed the successful repression of the target genes, along with few polar effects and differential knockdown level depending on PAM strength and target site. Both PG modifications were found to contribute to beta-lactam resistance. While the amidation of D--glutamate impacted cefotaxime and isoniazid resistance, the -glycolylation of muramic acid substantially promoted resistance to the tested beta-lactams. Their simultaneous depletion provoked synergistic reductions in beta-lactam MICs. Moreover, the depletion of these PG modifications promoted a significantly faster bacilli killing by J774 macrophages. Whole-genome sequencing revealed that these PG modifications are highly conserved in a set of 172 clinical strains of , demonstrating their potential as therapeutic targets against TB. Our results support the development of new therapeutic agents targeting these distinctive mycobacterial PG modifications.

CRISPRi-mediated characterization of novel anti-tuberculosis targets: Mycobacterial peptidoglycan modifications promote beta-lactam resistance and intracellular survival.

The golden age of antibiotics for tuberculosis (TB) is marked by its success in the 1950s of the last century. However, TB is not under control, and the rise in antibiotic resistance worldwide is a major threat to global health care. Understanding the complex interactions between TB bacilli and their host can inform the rational design of better TB therapeutics, including vaccines, new antibiotics, and host-directed therapies. We recently demonstrated that the modulation of cystatin C in human macrophages via RNA silencing improved the anti-mycobacterial immune responses to  infection. Available in vitro transfection methods are not suitable for the clinical translation of host-cell RNA silencing. To overcome this limitation, we developed different RNA delivery systems (DSs) that target human macrophages. Human peripheral blood-derived macrophages and THP1 cells are difficult to transfect using available methods. In this work, a new potential nanomedicine based on chitosan (CS-DS) was efficiently developed to carry a siRNA-targeting cystatin C to the infected macrophage models. Consequently, an effective impact on the intracellular survival/replication of TB bacilli, including drug-resistant clinical strains, was observed. Altogether, these results suggest the potential use of CS-DS in adjunctive therapy for TB in combination or not with antibiotics.

Development of Chitosan Particles Loaded with siRNA for Cystatin C to Control Intracellular Drug-Resistant .

Human immunodeficiency virus (HIV) and  (Mtb) are pathogens responsible for millions of new infections each year; together, they cause high morbidity and mortality worldwide. In addition, late-stage HIV infection increases the risk of developing tuberculosis (TB) by a factor of 20 in latently infected people, and even patients with controlled HIV infection on antiretroviral therapy (ART) have a fourfold increased risk of developing TB. Conversely, Mtb infection exacerbates HIV pathogenesis and increases the rate of AIDS progression. In this review, we discuss this reciprocal amplification of HIV/Mtb coinfection and how they influence each other's pathogenesis. Elucidating the infectious cofactors that impact on pathogenesis may open doors for the design of new potential therapeutic strategies to control disease progression, especially in contexts where vaccines or the sterile clearance of pathogens are not effectively available.

HIV/Mtb Co-Infection: From the Amplification of Disease Pathogenesis to an "Emerging Syndemic".

Esters of weak acids have shown improved antimycobacterial activity over the corresponding free acids and nitro benzoates in particular have previously shown to have a very intriguing activity. To expand the potential of nitro-derivatives of benzoic acid as antimycobacterial drugs and explore the effects of various structural features on the activity of these compounds, we have obtained a library of 64 derivatives containing esters and thioesters of benzoates and studied their activity against , the stability of the compounds, their activation by mycobacterial enzymes and the potential cytotoxicity against human monocytic THP-1 cell line. Our results showed that the most active compounds are those with an aromatic nitro substitution, with the 3,5-dinitro esters series being the most active. Also, the greater antitubercular activity for the nitro derivatives was shown to be unrelated to their pKa values or hydrolysis rates. Given the conventional relationship between nitro-containing substances and toxicity, one might anticipate that the great antimicrobial activity of nitro compounds would be associated with high toxicity; yet, we have not found such a relationship. The nitrobenzoate scaffold, particularly the 3,5-dinitrobenzoate scaffold, merits further investigation, because it has the potential to generate future antimycobacterial agents with improved activity.

Nitrobenzoates and Nitrothiobenzoates with Activity against .

Macrophages (Mø) and dendritic cells (DCs) are key players in human immunodeficiency virus (HIV) infection and pathogenesis. They are essential for the spread of HIV to CD4+ T lymphocytes (TCD4+) during acute infection. In addition, they constitute a persistently infected reservoir in which viral production is maintained for long periods of time during chronic infection. Defining how HIV interacts with these cells remains a critical area of research to elucidate the pathogenic mechanisms of acute spread and sustained chronic infection and transmission. To address this issue, we analyzed a panel of phenotypically distinct HIV-1 and HIV-2 primary isolates for the efficiency with which they are transferred from infected DCs or Mø to TCD4+. Our results show that infected Mø and DCs spread the virus to TCD4+ via cell-free viral particles in addition to other alternative pathways. We demonstrate that the production of infectious viral particles is induced by the co-culture of different cell populations, indicating that the contribution of cell signaling driven by cell-to-cell contact is a trigger for viral replication. The results obtained do not correlate with the phenotypic characteristics of the HIV isolates, namely their co-receptor usage, nor do we find significant differences between HIV-1 and HIV-2 in terms of cis- or trans-infection. The data presented here may help to further elucidate the cell-to-cell spread of HIV and its importance in HIV pathogenesis. Ultimately, this knowledge is critical for new therapeutic and vaccine approaches.

Cell-to-Cell Transmission of HIV-1 and HIV-2 from Infected Macrophages and Dendritic Cells to CD4+ T Lymphocytes.

 (Mtb), the causative agent of human tuberculosis (TB), is one of the most successfully adapted human pathogens. Human-to-human transmission occurs at high rates through aerosols containing bacteria, but the pathogen evolved prior to the establishment of crowded populations. Mtb has developed a particular strategy to ensure persistence in the host until an opportunity for transmission arises. It has refined its lifestyle to obviate the need for virulence factors such as capsules, flagella, pili, or toxins to circumvent mucosal barriers. Instead, the pathogen uses host macrophages, where it establishes intracellular niches for its migration into the lung parenchyma and other tissues and for the induction of long-lived latency in granulomas. Finally, at the end of the infection cycle, Mtb induces necrotic cell death in macrophages to escape to the extracellular milieu and instructs a strong inflammatory response that is required for the progression from latency to disease and transmission. Common to all these events is ESAT-6, one of the major virulence factors secreted by the pathogen. This narrative review highlights the recent advances in understanding the role of ESAT-6 in hijacking macrophage function to establish successful infection and transmission and its use as a target for the development of diagnostic tools and vaccines.

David Pires

Faculty of Medicine,
Center for Interdisciplinary Research in Health

Universidade Católica Portuguesa

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

David Pires

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Faculty of Medicine,Center for Interdisciplinary Research in Health

Universidade Católica Portuguesa

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Faculty of Medicine,
Center for Interdisciplinary Research in Health