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By Susha Cheriyedath, M.Sc. Jul 31 2023 Reviewed by Benedette Cuffari, M.Sc.

A recent study published in PLOS Pathogens identifies the Leptospira membrane protein L36 (MPL36) as essential for its pathogenesis and, as a result, a potential target for future diagnostics and prophylactics.

Study:  MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira, has an essential role during infection.  Image Credit: Kateryna Kon / Shutterstock.com Background

The human plasminogen (PLG) system provides a proteolytic substrate for pathogen invasiveness and tissue damage, thereby increasing the virulence of various bacterial species. Sequestration of PLG and activation into plasmin (PLA) is important for bacterial survival, as PLA degrades fibrin clots, extracellular matrix (ECM) molecules, and the host's innate immunological proteins to enable bacteria to spread and evade immune responses ultimately.

Leptospirosis, a life-threatening infectious disease, is a zoonotic disease that accounts for over one million infections and about 60,000 deaths each year throughout the world. In severe forms of leptospirosis, patients will often experience severe hemorrhaging and acute kidney injury.

Leptospira enter the host through injured skin or mucous membranes and subsequently travel through viscous environments to ultimately reach the bloodstream. Thereafter, Leptospira participate in cellular adhesion, which is mediated by various surface proteins that interact with ECM components.

Previous studies indicate that many Leptospira proteins act as PLG receptors, some of which include outer membrane proteins (OMPs) like endostatin-like protein A (LenA), as well as Leptospiral immunoglobulin-like proteins A (LigA), LiGB, and LipL32.

To date, there remains a lack of data on the pathogenesis of leptospirosis, which has prevented the development of sensitive diagnostic instruments and efficient prophylactic treatments. The present study reports that MPL36 binds with PLG, promotes adherence to host cells, and degrades fibrinogen by converting bound PLG to PLA. About the study

The current study utilized Leptospira interrogans serovar Manilae strain L495 and its mutant stains, including Manilae mpl36 mutant complemented strain (Δmpl36+) and Leptospira biflexa serovar Patoc strain Patoc 1. These strains were cultured in Madin-Darby canine kidney (MDCK) cells, following which their growth curves were analyzed using dark-field microscopy, motility, and immunoblot assays.  

Recombinant MPL36 (rMPL36) protein was produced using E. coli expression. Intrinsic tryptophan fluorescence spectroscopy and nickel affinity chromatography evaluated the tertiary structure of rMPL36, rMPL36 aa41-305, and rMPL36 aa41-235. The binding capacity of rMPL36 was determined, and the conversion of PLG bound to rMPL36 into active PLA was evaluated.

The binding of rMPL36 to ECM proteins and PLG was determined by staining for fibronectin, laminin, PLG, and bovine serum albumin (BSA). The pathogenicity of Manilae wildtype (WT) and mutant strains was also evaluated in three-week-old male Golden Syrian hamsters. Related StoriesSARS-CoV-2 spike protein's role in lung injury unraveled: promising new approach uncoveredMitochondrial repair protein discovery may lead to new anti-aging treatmentsSalk researchers uncover molecular mechanisms of HIV drug resistance

The tertiary structure of sporulation-related repeat (SPOR) domains from leptospiral species was predicted using AlphaFold and PyMOL software ClustalX to match MPL36 SPOR domain sequences from various Leptospira species. In vivo data was analyzed using Prism 9, Fisher's exact test, and analysis of variance (ANOVA) to ecludate the mechanisms of rMPL36 binding to host components and BSA. MPL36 is a crucial target for leptopsirosis

The motility assay revealed that, as compared to wild-type cells, mpl36 mutant and complemented strains did not significantly differ in their motility or growth characteristics. Immunofluorescence staining confirmed the subcellular localization of MPL36 in Leptospira as an outer membrane protein in both wild-type and complement cells.

Immunoprecipitation studies revealed that rMPL36 exhibited significant binding to both rLigA and rLigB, whereas no binding was observed between rMPL36 and fibronectin nor laminin.

However, rMPL36 was found to effectively bind to human PLG as compared to other recombinant proteins that were used as positive and negative controls. Within rMPL36, the conserved SPOR domain, which is located at the last 70 amino acids of the protein, is essential for the binding between rMPL36 and PLG.

Hamsters that were infected with the wild-type strain died between eight and nine days following infection with Leptospira. Comparatively, mpl36 mutant hamsters who were infected with 108 leptospires survived without exhibiting any symptoms of leptospirosis, thereby demonstrating the significant role of MPL36 in leptospirosis.

Convalescent sera were obtained from Brazilian individuals with laboratory-confirmed severe leptospirosis. To this end, anti-MPL36 immunoglobulin M (IgM) and IgG were observed in leptospirosis patients’ samples as compared to sera from healthy individuals. Study takeaways

MPL36 is an outer membrane Leptospira protein that is capable of binding to PLG, degrading fibrinogen, and facilitating host infection. Despite these findings, additional research is needed to better understand leptospiral pathogenesis and advance diagnostic and prevention strategies. Journal reference: Zhu, W., Passalia, F. J., Hamond, C., et al. (2023). MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira, has an essential role during infection. PLOS Pathogens. doi:10.1371/journal.ppat.1011313 https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011313

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USAID crisis leaves South Africans living with HIV in turmoil

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USAID crisis leaves South Africans living with HIV in turmoil

A woman walks up to the security guards outside a shuttered USAID-funded sexual health clinic in Johannesburg’s inner-city district.

She looks around with confusion as they let her know the clinic is closed.

She tells us it has only been two months since she came here to receive her usual care.

Now, she must scramble to find another safe place for her sexual health screenings and Pre-Exposure Prophylaxis (PrEP) – her regular defence against rampant HIV.

On the day he was sworn in as US president for a second time, Donald Trump signed an executive order freezing foreign aid for a 90-day period.

That is being challenged by federal employee unions in court over what it says are “unconstitutional and illegal actions” that have created a “global humanitarian crisis”.

However the order is already having an immediate impact on South Africa’s most vulnerable.

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Her eyes tear up as she processes the news. Like many sex workers in town, free sexual health clinics are her lifeline.

An HIV-positive sex worker shared her patient transfer letter from the same closed clinic with Sky News and told us with panic that she is still waiting to be registered at an alternative facility.

South Africa is home to one of the world’s worst HIV/AIDS epidemics. At least 8.5 million people here are living with HIV – a quarter of all cases worldwide.

Widespread, free access to antiretroviral treatment in southern Africa was propelled by the introduction of George W. Bush’s US President Emergency Plan for AIDS Relief (PEPFAR) in 2003.

PEPFAR is considered one of the most successful foreign aid programmes in history, and South Africa is the largest recipient of its funds.

A sign for USAID on the clinic's window
Image:
A sign for USAID on the clinic’s window

A shuttered USAID-funded sexual health clinic in Johannesburg
Image:
A shuttered USAID-funded sexual health clinic in Johannesburg

The programme has now been halted by President Trump’s foreign aid funding freeze – plunging those who survived South Africa’s HIV epidemic and AIDS denialism in the early 2000s back to a time of scarcity and fear.

“That time, there was no medication. The government would tell us to take beetroot and garlic. It was very difficult for the government to give us treatment but we fought very hard to win this battle. Now, the challenge is that we are going back to the struggle,” says Nelly Zulu, an activist and mother living with HIV in Soweto.

Nelly says access to free treatment has saved her and her 21-year-old son, who tested positive for HIV at four years old.

“It helped me so much because if I didn’t get the treatment, I don’t think I would be alive – even my son.

“My concern is for pregnant women. I don’t want them to go through what I went through – the life I was facing before. I’m scared we will go back to that crisis.”

Nelly Zulu, an activist and mother living with HIV
Image:
Nelly Zulu, an activist and mother living with HIV

South African civil society organisations have written a joint open letter calling for their government to provide a coordinated response to address the healthcare emergency created by the US foreign aid freeze.

The letter states that close to a million patients living with HIV have been directly impacted by stop-work orders and that a recent waiver by US Secretary of State Marco Rubio continuing life-saving assistance explicitly excludes “activities that involve abortions, family planning, gender or diversity, equality and inclusion ideology programmes, transgender surgeries or other non-life saving assistance”.

The shuttered clinic we saw in Johannesburg’s central business district (CBD) comes under these categories – built by Witwatersrand University to research reproductive health and cater to vulnerable and marginalised communities.

An activist and healthcare worker at a transgender clinic tells us everyone she knows is utterly afraid.

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USAID in turmoil: What you need to know

“Corner to corner, you hear people talking about this. There are people living with chronic diseases who don’t have faith anymore because they don’t know where they are ending up,” says Ambrose, a healthcare worker and activist.

“People keep asking corner to corner – ‘why don’t you go here, why don’t you go there?’ People are crying – they want to be assisted.”

South Africa’s ministry of health insists that only 17% of all HIV/AIDs funding comes from PEPFAR but that statistic is offset by the palpable disruption.

On Monday, minister of health Dr Aaron Motsoaledi met to discuss bilateral health cooperation and new US policy for assistance with US charge d’affaires for South Africa, Dana Brown.

A statement following the meeting says: “Communication channels are open between the Ministry and the Embassy, and we continue to discuss our life-saving health partnership moving forward.

“Until details are available the minister called on all persons on antiretrovirals (ARVs) to under no circumstances stop this life-saving treatment.”

A demand much harder to execute than declare.

“There is already a shortage of the medication – even if you ask for three months’ treatment, they will give you one or two months worth then you have to go back,” says Nelly.

“Now, it is worse because you can see the funding has been cut off.”

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Politics

Utah takes the lead in potentially enacting a Bitcoin reserve bill

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Utah takes the lead in potentially enacting a Bitcoin reserve bill

A bill that would give the Utah treasurer the power to buy BTC and other high-cap crypto assets with public funds is on its way to the Senate.

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SEC acknowledges Grayscale Solana ETF filing in ‘notable’ step

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SEC acknowledges Grayscale Solana ETF filing in ‘notable’ step

It’s one more development reflecting a change in approach from the US Securities and Exchange Commission toward crypto.

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